- Methodology article
- Open Access
High-resolution ex vivo magnetic resonance angiography: a feasibility study on biological and medical tissues
© Rasmussen et al; licensee BioMed Central Ltd. 2010
Received: 3 July 2009
Accepted: 12 March 2010
Published: 12 March 2010
In biomedical sciences, ex vivo angiography is a practical mean to elucidate vascular structures three-dimensionally with simultaneous estimation of intravascular volume. The objectives of this study were to develop a magnetic resonance (MR) method for ex vivo angiography and to compare the findings with computed tomography (CT). To demonstrate the usefulness of this method, examples are provided from four different tissues and species: the human placenta, a rice field eel, a porcine heart and a turtle.
The optimal solution for ex vivo MR angiography (MRA) was a compound containing gelatine (0.05 g/mL), the CT contrast agent barium sulphate (0.43 mol/L) and the MR contrast agent gadoteric acid (2.5 mmol/L). It was possible to perform angiography on all specimens. We found that ex vivo MRA could only be performed on fresh tissue because formalin fixation makes the blood vessels permeable to the MR contrast agent.
Ex vivo MRA provides high-resolution images of fresh tissue and delineates fine structures that we were unable to visualise by CT. We found that MRA provided detailed information similar to or better than conventional CTA in its ability to visualize vessel configuration while avoiding interfering signals from adjacent bones. Interestingly, we found that vascular tissue becomes leaky when formalin-fixed, leading to increased permeability and extravascular leakage of MR contrast agent.
In vivo angiography is frequently employed to produce a structural overview of the intravascular configuration in living tissues  whereas angiography of excised organs or post-mortem angiography is rarely performed [2–4]. For example, ex vivo angiography has been used successfully to identify structural components of carotid atherosclerotic plaques  and to visualize the renal microvasculature . In addition, post mortem angiography provides information secondary to conventional autopsy in humans and animals [7, 8]. Computed tomography angiography (CTA) can be employed with sub-millimetre resolution of vessels in conjunction with an intravascular contrast agent . However, accurate delineation of the entire vessel configuration acquired by CTA is often complicated by the inherent absorption of X-rays in bones and cartilage, characterized as hyperintense areas on CTA images. Magnetic resonance angiography (MRA) is another method, where an intravascular confined paramagnetic compound generates a change in the local magnetization of water, resulting in a hyperintense signal of the streaming blood. MRA has potential advantages over CTA in its unique ability to reveal the intravascular compartment without concomitant contributions from bone and cartilage. On the other hand, the small molecules of most available magnetic resonance contrast agents leak rapidly into the interstitial compartment, hampering detailed and prolonged intravascular measurements using this technique. In clinical situations, this extravasation from the intravascular compartment is overcome by performing an MRA procedure in which images are acquired during the first pass of the agent through the arteries. However, as rapid circulation of blood in smaller species and the lack of circulation in excised organs and dead subjects preclude the use of this approach, there are today no available MRA techniques allowing three-dimensional (3D) high-resolution images of blood vessels in these situations.
This methodological study presents a novel method for ex vivo MRA. To demonstrate the usefulness of this method, examples of both zoological and biomedical relevance were studied from four distinctly different tissues: the human placenta, a rice field eel, a porcine heart and a turtle.
Results and Discussion
The experimental study was conducted in several steps. Prior to MRA and CTA of selected specimens, we defined the optimal concentration of contrast agents. Next, the use of formalin-fixed and fresh specimens was elucidated to reveal the microstructural consequences of protein binding in the presence of formalin. Three different specimens were then subjected to the chosen MRA and CTA protocols. First, fresh placentas were harvested following elective caesarean section from our obstetric department. Second, live specimens of rice field eel (Monopterus albus) were purchased from aquacultures in Vietnam. They were kept in a 500 L plastic aquarium in a 12:12 h L:D routine, kept at 29°C and fed with mussels and shrimps. Third, excised hearts were used from healthy female Danish Landrace pigs weighing 65 kg. Fourth, turtles (Trachemys scripta) were examined to demonstrate the proposed MRA and CTA methods on a complete animal.
Preparation of contrast agent
2. Two different contrast agent solutions were prepared. For contrast mix 1, saline was heated to 60°C and gelatine at a final concentration of 0.05 g/mL (gelatine for microbiology, Merck, Darmstadt, Germany) was added in small portions while stirring together with the CT contrast agent barium sulphate at a final concentration of 0.43 mol/L (Mixobar Colon, 1 g/mL; Astra Tech, Sweden). Dotarem was added to a final concentration of 2.5 mmol/L when the solution had cooled to 40°C. Contrast mix 2 contained saline and the following ingredients: the MR contrast agent gadofosveset trisodium (Vasovist; Bayer-Schering Pharma, Germany) at 2.5 mmol/L and albumin at 60 g/L (egg white albumin; Sigma-Aldrich, USA). The saline was heated to 40°C, and albumin and Vasovist were added while stirring. As Vasovist binds to albumin, the gadolinium-containing compound cannot diffuse from the intra- to the extravascular compartment and Vasovist is therefore considered a suitable intravascular contrast agent for living specimens. Additionally, a commercially made CT contrast agent, Microfil (Flow Tech, USA), was used for CTA of the rice field eel head, which was compared with the acquired MRA .
Comparing results obtained with contrast mixes 1 and 2 (data not shown) demonstrated that contrast mix 1 was better by serving both as an MRA and a CTA agent. This property facilitates consecutive MRA and CTA on the same sample. However, contrast mix 2 may be of benefit thanks to its lower viscosity and easier distribution to small capillary vessels.
Microstructural effect of formalin fixation
We have demonstrated a novel experimental method to generate a 3D representation of blood vessels using MRA specifically applied to ex-vivo studies. We found that MRA provided detailed information similar to or better than conventional CTA. MRA has a great advantage over CTA in its ability to visualize only the vessel configuration without interfering signals from adjacent bones as seen in CTA. In all cases, we found that the best method to obtain high-quality ex vivo MRA was to perfuse the blood vessels with saline containing heparin at 40°C immediately after organ harvesting, then lowered into a water-bath at 40°C and perfused with contrast solution. In cases where both MRA and CTA must be performed on the same specimen, contrast mix 1 is to be preferred whereas in cases where only MR scans are performed contrast mix 2 is preferable. It was observed that placental vascular tissue becomes leaky when formalin-fixed, leading to increased permeability and outward diffusion of MR contrast agent.
This study complied with the Helsinki Declaration. Approval to use human placentas was given by the The Danish National Committee on Biomedical Research Ethics (approval #M-20080152), and all pregnant woman gave their written consent for the use of their placenta. Approval to include animals in this study was given by the The Danish Ministry of Justice for Animal Experiments Inspectorate (approval #2008-561-1522).
MRA and CTA
MRA was performed with clinically available MR systems, a GE Horizon Echospeed LX 1.5 T (GE Healthcare, United Kingdom) and a Philips Achieva 1.5 T (Philips Medical Systems, Netherlands). Each specimen was positioned in a quadrature radiofrequency head coil. A fast localizer scan was followed by a high-resolution 3D gradient-echo sequence with the following parameters: field-of-view depending on specimen; thickness 1 mm; TR 23.1 ms; TE 1.6 ms and excitation flip angle 30°. A stack of multiple slices (with no gaps) was acquired, covering the entire specimen of interest with scan durations of 30-60 min. Image resolution was 0.5 × 0.5 × 0.5 mm3
CTA was performed using a 64-slice Siemens Somatom Definition (Siemens Medical Solutions, Germany) with dual source capacity. Acquisition parameters included a slice collimation of 4 mm; a pitch of 2°; 32 rotations (resulting in a 25.6 cm scanning volume) and a matrix size of 512 × 512. Acquisition parameters using the Somatom Volume Zoom option included a slice collimation of 4 × 1 mm, a rotation time of 0.5 s, 5-8 mm table feed/rotation, a matrix size of 512 × 512 and scan duration of 25-30 s. Transverse images were reconstructed with a section thickness of 1.25 mm and were reconstructed at 0.6 mm intervals.
Data acquired both by MRA and CTA were exported to DICOM format and a 3D reconstruction was performed using either the Mistar http://www.apollomit.com or the Osirix software http://www.osirix-viewer.com, allowing a maximum-intensity-projection in arbitrary directions.
Two tissue samples were withdrawn from fresh placental blood vessels and two from formalin-fixated placenta blood vessels. The samples were freeze-dried and mounted on sample holders. The sample holders were placed in an Edward's gold sputter-coater, which covered the sample surface with a thin layer of gold. Subsequently, the samples were placed in a CamScan MaXim 2040 EnVac SEM (CamScan, United Kingdom) and images were obtained using secondary electrons and an Everhart-Thornley detector.
We thank Jesper Thygesen for valuable help during CTA procedures. Troels Thim helped in making the contrast solutions and we thank Jacques Chevalier for SEM assistance.
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