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Original Research |
1 Institute of Forensic Medicine, University of Bern, IRM, Buehlstrasse 20,
CH-3012 Bern, Switzerland.
2 Institute of Anatomy, University of Bern, Bern, Switzerland.
3 Institute of Veterinary Anatomy, University of Bern, Bern, Switzerland.
4 CT/MRI-Center, Graz, Austria.
5 Institute of Diagnostic Radiology, University of Bern, Bern,
Switzerland.
Received August 10, 2005;
accepted after revision September 29, 2005.
Supported by the Virtopsy Foundation and the Swiss National Science
Foundation (3100AQ-104000/1).
Abstract
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MATERIALS AND METHODS. Cadavers of two dogs and one cat were perfused with diesel oil through a peristaltic pump. The lipophilic contrast agent Lipiodol Ultra Fluide was then injected, and angiography was performed. The efficiency of perfusion was evaluated in the chick chorioallantoic membrane.
RESULTS. Vessels could be seen up to the level of the smaller supplying and draining vessels. Hence, both the arterial and the venous sides of the vascular system could be distinguished. The chorioallantoic membrane assay revealed that diesel oil enters microvessels up to 50 µm in diameter and that it does not penetrate the capillary network.
CONCLUSION. After establishing a postmortem circulation by diesel oil perfusion, angiography can be performed by injection of Lipiodol Ultra Fluide. The resolution of the images obtained up to 3 days after death is comparable to that achieved in clinical angiography.
Keywords: angiography • animal studies • contrast media • noninvasive autopsy • postmortem angiography • virtopsy • virtual autopsy
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The techniques that have been applied for postmortem angiography are various and have been comprehensively reviewed by Schoenmackers [5] and Grabherr [6]. The perfusates used for postmortem angiography can be broadly divided into two categories—namely, hydrophobic fluids and aqueous media. The former are confined to the intravascular space, whereas the latter extravasate and penetrate into the surrounding tissues [7]. Aqueous media, which are more penetrating than hydrophobic fluids, are used to embalm cadavers [8]. As the interval since death increases, the process of extravasation becomes very pronounced.
Postmortem angiography is of use mainly in forensic medicine and in pathoanatomy [9]. The technique has been applied to show coronary [10] and splenic [11] arteries and to analyze traumatically ruptured bridging veins in the brain [12].
We have elaborated a new minimally invasive technique for postmortem angiography using an oily perfusate and a lipophilic contrast agent. The approach involves two steps. Initially, postmortem circulation is established by perfusing the vascular system with an oily fluid [13] through a peristaltic pump [14]. The vascular system is then rendered visible by injecting a lipophilic agent into the established circulation. Diesel oil was chosen for the perfusate because of its low viscosity, and Lipiodol Ultra Fluide (ethyl ester of iodized poppy seed oil, Guerbet AG) [15] was used as the contrast agent.
A feasibility study was first conducted using the cadavers of two dogs and a cat. The hemodynamics of the system were then evaluated in the living chorioallantoic membrane of a chicken.
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For the caudal circulation, a similar procedure was performed in the feline cadaver. In this case, the polytetrafluoroethylene tubes were inserted into the abdominal aorta and inferior vena cava in a cranial-to-caudal direction. The vessels were ligated cranially.
For the whole-body circulation in the second canine cadaver, the left common carotid artery and the left internal jugular vein were cannulated. The polytetrafluoroethylene tubes were inserted in a caudal-to-cranial direction.
After these preparations, each cannula was connected to a peristaltic pump (HR-Flow-inducer, Watson-Marlow Bredel). The pump was filled with diesel oil containing Sudan red III (0.01%). The vascular system was perfused for 5-10 minutes. The cadavers were then transported to the radiology laboratory.
Angiography—For visualization of the cranial circulation, angiography with conventional radiography equipment (Cygnus Rad 35, Odel) was performed; each cadaver was placed in a custom-made plastic box (Semadeni) together with the peristaltic pump. Perfusion with diesel oil was reinitiated, and two native radiographs were obtained while the system was running. Forty milliliters of iodized oil (Lipiodol Ultra Fluide, Guerbet AG) was then injected immediately using a membrane applicator built into the tube system of the pump. During maintained perfusion, four angiographic projections were taken within an 8-minute period and three more at intervals of 20 minutes. The perfusion was then stopped, and the plastic box containing the cadaver and the pump was transported to the MDCT unit (Somatom Sensation 16, Siemens Medical Solutions).
Perfusion was reinitiated, and the first MDCT scan was obtained while the system was running. Scanning parameters were 0.75-mm collimation, 0.75-mm slice width, and reconstruction increment of 0.7 mm. Forty milliliters of Lipiodol Ultra Fluide were then injected immediately using the membrane applicator. Nine scans were obtained within 1 hour during continuous perfusion.
For caudal and whole-body angiography, these cadavers (one cat and one dog) were subjected only to MDCT scanning, which was performed as described. In the cat (posterior circulation), nine scans were obtained within 1 hour. In the second dog (whole-body circulation), seven scans were obtained over a similar time period.
Data evaluation—The angiograms were evaluated by two board-certified radiologists who were not present when the experiments were performed and by a veterinary anatomist.
Chorioallantoic Membrane Study
Culturing of chicken embryos—The shell-free culturing method
described by Auerbach et al.
[16] in 1974 was used in this
study. After 3 days of incubation, the eggs were opened and the contents were
transferred to a plastic Petri dish. The embryos were incubated for 12 days at
37°C. On day 10, they were divided into three groups.
In group 1, the vasculature of the embryos was injected first with 0.1 mL of 2.5% aqueous solution of fluorescein isothiocyanate dextran (FITC) 1000000 (FD-1000S, Sigma) and then with 0.5 mL of diesel oil, under microscopic control. Because the two fluids do not mix, they were filled into the same 1-mL syringe and injected sequentially via a 0.8-mm-diameter needle.
In group 2, the vasculature of the embryos was injected first with 0.5 mL of diesel oil and then with 0.1 mL of aqueous FITC under microscopic control.
In group 3, the vasculature of the embryos was injected first with 0.5 mL of an emulsion of sodium fluoride (Fluorol, GAF GmbH) and diesel oil under microscopic control.
Fluorescence microscopy—The vascular injection process and the distribution of the fluids were recorded using a video camera (LE CCD, Optronics) equipped with a digital video recorder (DHR-1000VC, Sony) that were attached to a fluorescence microscope (Polyvar, Reichert). The vessels were inspected in the same microscope using x10 and x25 objectives.
Data evaluation—Microscopic evaluation was performed by an anatomist who specializes in the field of chorioallantoic membrane assay and vascular biology.
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The first vessel to appear was the facial vein. After 8 minutes, the entire venous system was visible. At this juncture, the arterial vessels were barely recognizable. On the last three angiograms, obtained 28, 48, and 68 minutes after Lipiodol injection, the contrast agent was increasingly washed out. Only the cardiac cavities and a few large vessels, such as the jugular and brachial veins, were patchily visible.
On the first MDCT angiography scan, the residual effects of the contrast agent used for conventional angiography were still apparent. The first two scans obtained 2 and 4 minutes after Lipiodol injection revealed the arterial phase of angiography (Figs. 1C and 1D). In addition to the vasculature that was visible on conventional angiography, the vessels of the forelimb and the ear were apparent in 3D reconstructions of the MDCT scans (Fig. 1D).
The venous side of the circulation was detected on the third scan, obtained 9 minutes after Lipiodol injection (Fig. 1E). At this juncture, the jugular and the cephalic veins, together with their branches, were visible. Fourteen minutes after Lipiodol injection (Fig. 1F), the venous phase was apparent. However, after 58 minutes, the scan was similar to the initial native scan.
Caudal feline circulation—MDCT angiograms obtained 2 (Figs. 2A and 2B) and 4 minutes after Lipiodol injection revealed the arterial side of the circulation. Vessels of the pelvis—for example, the inferior epigastric artery and the internal iliac artery—and of the thigh—for example, the common, deep, and superficial femoral arteries and their branches— were revealed on both 3D reconstructions (Fig. 2A) and cross sections (Fig. 2B). On the third scan, taken 9 minutes after Lipiodol injection (Fig. 2C), the effects of the contrast agent had begun to fade. Twenty-five minutes after Lipiodol injection (Fig. 2D), only the largest veins, such as the medial saphenous and femoral veins, were visible. After 58 minutes (Fig. 2E), the entire vascular system had been cleared of the contrast agent. Only the left saphenous vein was patchily disclosed. It is noteworthy that on all MDCT angiograms, Lipiodol did not penetrate beyond the region of the talocalcaneal joint.
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Postmortem angiography represents one minimally invasive autopsy tool. Using classic autopsy methods, the vascular system is not readily shown. Angiography should permit the detection of vessel abnormalities and injuries. In combination with imaging techniques, postmortem angiography should render possible visualization of aneurysms, ruptured aneurysms, and vascular tears such as those incurred by the intercostal arteries after fracturing of the ribs that are not easily discerned at a conventional autopsy. Angiography will also permit the detection of small intraparenchymatous bleeding, knowledge of which is important for the reconstruction of traumas. Furthermore, we anticipate that postmortem angiography will have an impact on the diagnosis of tumors and of circulation disorders.
Until now, optimal postmortem visualization of the vascular system has not been possible, owing to the increasing permeability of the vascular wall after death. Diverse attempts have been made to improve the resolution for diagnostic purposes [6]. During the past few decades, the use of oily perfusates has declined, even though these were shown in the 1970s to be retained intravascularly for at least the first 72 hours after death [7]. We have now pursued this early finding. Conceptually, combining an oily perfusate with a lipophilic contrast agent permits the establishment of postmortem circulation and the performance of subsequent high-resolution angiography up to 3 days after death.
In our experiments, the perfusate entered the venous system after 1-2 minutes. It was thus a moot point whether the parenchymatous phase, which involves a discharge of the capillaries, was restricted or not. To answer this question, we undertook a microscopic study of chorioallantoic membranes. Our findings revealed that the oil blocks this region, which is especially vulnerable to postmortem permeability.
The oil entered the venous system by passing through small arteriovenous shunts while the capillary microcirculation was arrested. Oily perfusates thus appear to be highly suitable for postmortem angiography. Our findings coincide with observations relating to the splitting of masses from their blood supply by chemoembolization [17]. A stoppage of the lipophilic contrast agent Lipiodol at the level of the arterioles has also been reported by Kan [18]. After injecting Lipiodol into the liver, Kan observed that it failed to penetrate the surrounding tissues from the sinusoidal region, where it was resorbed by phagocytes.
The vascular resolution that can be achieved with Lipiodol is remarkable. The high contrast of the images obtained is attributable to the high density of this agent, which is twice that of bone. This high density can also generate artifacts, but these artifacts can be minimized by lowering the concentration of the agent. The rinsing out of postmortem blood clots is another advantage of an oily perfusate. After intraarterial application, it permits additional visualization of the venous side of the vascular system [19].
Visualization of the coronary vessels and of the peripheral vessels of the extremities well enough to allow diagnosis is still problematic. The unsatisfactory results achieved in these regions may reflect both the positioning of the animals and the uncontrolled injection pressure of Lipiodol. Jackowski et al. [2] have recently shown that these problems can be avoided by controlling the injection pressure. We are now in the process of developing a pressure-controlled pump that is analogous to the heart-lung machine that has already been established. Initial experiments with human cadavers have yielded promising results.
The strong odor of diesel oil is a disadvantage of this perfusate. A less odoriferous alternative is paraffin oil [13].
Our experiments revealed a clear locus minoris resistentiae in the gastrointestinal tract. In this region, an articulate dehiscence of the vascular wall appears to take place shortly after death, and this change leads to a discharge of the perfusate into the stomach and the intestine. This finding is not surprising given the combination of bacterial decomposition and autolytic activities that occurs in this region and that leads to an increase in vascular permeability earlier in this region than in other body parts. This problem remains a challenge to be resolved in future investigations.
In conclusion, after establishing a postmortem circulation by perfusing the vascular system with diesel oil, angiography can be performed after the injection of a lipophilic contrast agent. The resolution of the vascular system achieved up to 3 days after death is comparable to that obtained in clinical angiography. Vessels can be seen up to the level of the smaller supplying and draining components of the vascular system. Hence, both the arterial and the venous sides can be distinguished. The vascular system can be seen without extravasation artifacts, tissue edema, or a background staining of the capillary plexus.
Near-optimal postmortem visualization of the vascular system opens new diagnostic possibilities in forensic medicine and pathology.
Acknowledgments
We gratefully acknowledge technical support from Kathrin Yen, Ruslan
Hlushchuck, Barbara Krieger, Gabriel von Almen, Emin Aghayev, Christian
Jackowski, Martin Sonnenschein, Roland Dorn, Urs Königsdorfer, Therese
Périnat, and Michael Stoffel.
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  S. Grabherr, V. Djonov, K. Yen, M. J. Thali, and R. Dirnhofer Postmortem Angiography: Review of Former and Current Methods Am. J. Roentgenol., March 1, 2007; 188(3): 832 - 838. [Abstract] [Full Text] [PDF]
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