The goal of this technique of contrast enhanced vessel imaging using microscopic computed tomography or micro CT is to provide an optimal method for generating high quality data sets for analysis of vascular networks in live mice. This is accomplished by intravenous injection of a radio opaque contrast agent in vivo. Then the animal is prepared for micro CT imaging by inducing anesthesia and positioning the animal securely in a multimodality chamber to reduce motion artifacts during micro CT scans.
Following scans, the micro CT data is reconstructed and loaded into the visualization software. The final step of the procedure is to adjust two dimensional transfer function parameters to improve the visibility of blood vessels and to provide different colors to blood vessels, bone and soft tissue for distinction purposes. Ultimately, results can be obtained that show the vascular network in live animals through CT opaque contrast enhanced imaging and two dimensional transfer function rendering.
Hi, I'm Sue Resh, PJA Patti from Small Animal Imaging Core at the Department of Khe Children's Cancer Research Institute at the University of Texas Health Science Center, San Antonio. I'm Charles Keller, also from the Greehey Children's Cancer Research Institute at the University of Texas Health Science Center in San Antonio. Today we will show you a procedure for visualization of blood vessels in live animals using radio opaque blood pool contrast agent.
We use this procedure in our laboratories to study the intermediate and large vessels of complex spontaneous tumors in our transgenic mice. So let's get started. To begin this procedure, obtain 0.2 milliliters per 20 grams of 20 milligrams of iodine per milliliter of iodinated blood pool Contrast agent Xia one 60 xl using a low dead space 28 and a half gauge half cc insulin syringe.
Inject the contrast agent into a live mouse via the distal tail vein. 10 minutes after the injection. Prepare the animals for micro CT imaging.
First, anesthetize the animals in an acrylic box connected to 2%isof fluorine in 100%oxygen at 2.5 liters per minute. After a few minutes, perform a toe pinch to verify the depth of anesthesia. Next to minimize movement artifacts, stabilize the mouse by placing it into a custom built, commercially available multimodality chamber with provision for air and exhaust.
Maintain an air supply with 2%isof fluorine and 100%oxygen to ensure continuous anesthesia of the animal during the scan. Once the mouse is prepared, it can be scanned on a micro CT unit capable of performing live animal scans. Connect the multimodality chamber containing the mouse to the errand exhaust tubes.
Using the volumetric CT scanner GE Explorer locus, the animal can be scanned at 93 micrometer resolution. This volumetric scanner uses a 3, 500 by 1750 CCD detector for Feld Camp cone beam reconstruction set the platform independent parameters constant with a current of 450 microamps, a voltage of a DP kilo voltage and an exposure time of 100 milliseconds. To achieve a superior contrast between bone and soft tissue from the vessel, standard parameters can be varied with five to eight frames per view and 360 to 720 number of views for a full 360 degree rotation, use a total scan time of approximately 15 to 20 minutes following CT scan.
Reconstruct images with the manufacturer's proprietary EVS beam software. Continue to monitor the mouse until it is fully recovered from anesthesia. Once reconstructed, the micro CT data can be used for advanced isosurface one dimensional or two dimensional transfer function.
Rendering images using SEG 3D image processing software to render a two dimensional projection of the three dimensional dataset, use a two dimensional transfer function which further improves the distinction between vessel and bone. During image rendering, assign colors and opacities to the ray sample based on the transfer function, which in turn relies on CT value and gradient magnitude. Manually adjust transfer functions based on guidance provided by CT value histograms to highlight areas and objects of interest shown.
Here are representative results showing a two dimensional transfer function rendering image on a micro CT scan of a wild type mouse injected with Xia one 60 xl. We've just shown you how to generate high quality data sets for analysis of vascular networks in live mice. This technique could very well be used for qualitative and quantitative assessment of anti-angiogenesis in preclinical studies using either transgenic or xenograft mice.
When doing this procedure, it's important to remember to get a perfect tail vein injection of the contrast agent and to position the animal in a way to minimize the motion artifact that can occur due to breathing. So that's it. Thanks for watching and good luck with your experiments.
