This method could help answer key questions in the vascular biology field, such as determining how vessel size is impacted in different animal models of vascular disease. The main advantage of this technique is that micro-CT enables detailed images of both large and small vessels, both in vivo and ex vivo, which are able to be precisely quantified. 24 hours prior to the CT analysis, perform the surgical procedures to prepare the specimen for a postmortem intracardiac profusion.
For anesthesia, use 3%isoflurane, and be sure to have charcoal scavenger in use. Then, maintain the anesthesia with two to 2.5 percent isoflurane from a face mask, also attached to a scavenger with one liter of oxygen per minute. Prior to beginning the surgery, perform a toe pinch to check for the absence of a pedal reflex.
Then, set up an operative field with instruments and so forth. Next, position the animal in dorsal recumbency and secure the limbs. Now, using scissors, make a midline incision through the skin and soft tissue over the sternum between the pubic symphysis to the sternal notch.
Next, use the scissors to create a hole in the diaphragm at the xiphoid process to enter the thoracic cavity. Then, continue using the scissors to dissect the diaphragm of the ventral chest wall bilaterally. Cut through the costal cartilages to separate the ribs from the sternum at the right sternal border.
Now, apply a fine hemostatic clamp to the tip of the sternum near the xiphoid process and move the hemostat cranially so that it is positioned over the head of the mouse. Then, sharply dissect any attachments between the heart and the chest wall. Limit the number of vessels damaged during the animal preparation, as this will cause the contrast agent to leak out of the severed vessels, which will change the volume required for a complete filling, and will introduce artifacts to the final imaging.
Now, connect a 27 gauge IV catheter needle to a syringe preloaded with 10 milliliters of heparinized PBS and fill all the tubing with the buffer, removing all of the air bubbles. Removal of all bubbles from the lines is essential. Then, immediately incise the right ventricle, or the inferior vena cava, to drain the heparin solution and blood.
To proceed, use the cannula to puncture the left ventricle, stabilized by the clamp. Conduct the profusion at a constant two milliliters per minute using a syringe pump. There should be visible blanching of the organs.
In a few minutes, the vena's circulation should be free of blood. Then, stop the pump and disconnect the catheter tubing from the syringe, but do not move the catheter needle. Now, separate the contrast agent solution into five milliliter aliquots, add the curing agent and mix well.
Then, load five milliliters of the mixture into a 10 milliliter syringe and profuse the animal with the contrast agent at a steady two milliliter per minute. Keep the infusion going past the point of seeing the casting agent exiting the animal. The contrast agent will cure in 20 minutes.
Once cured, harvest the organs of interest and fix them in 10%neutral buffered formalin. Next, it the samples are not used for micro-CT scanning, fix the whole carcass. Otherwise, position the carcass on a metal tray and place it in a refrigerator at four degrees Celsius to cure overnight.
On the following day, acquire x-ray computed tomography images of each mouse using a micro-CT scanner. Set the scanner parameters as follows;An x-ray tube peak voltage of 55 kilovolts, a current of 150 microamps, a system magnification factor of 2.19, and a CCD camera pixel binning factor of two. This binning factor will yield an effective pixel size of 29 microns.
Next, analyze the images to identify aneurysms using a standardized approach. Measure the minor axis diameter at the widest point of three locations;of the aortic arch, the descending thoracic aorta, and the abdominal aorta. Ideally, two observers, one of which is blinded, should analyze each image.
Using the measurements, identify aneurysms as localized or diffused dilations of the aortic segments to diameters greater than 50%of the reference diameter. 20 male adult mice of mixed background and 20 to 30 weeks old were analyzed with or without BAPN treatment. Each was profused with a lead-based radiopaque silicone rubber.
The mean diameter of the ascending aorta in the BAPN-treated mice was significantly larger than that of the untreated age-matched controls. Data points from mice determined to have aneurysms are in red. By contrast, the inhibition of lysyl oxidase with BAPN did not have a significant effect on the mean descending thoracic, nor did this treatment affect abdominal aortic diameters.
An aneurysm was defined as being 1.5 times the mean diameter of the untreated group. The treatment lead to a significant increase in aneurysms by this definition. Aneurysms in the BAPN-treated mice were identified exclusively in the aorta, with the majority identified in the thoracic aorta.
After watching this video, you should have a good understanding of how to profuse the vasculature for radiographic imaging. Once mastered, the profusion and image analysis can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember that CT imaging protocols can be adjusted with the assistance of radiologic staff to optimize image acquisition for the specific purposes of individual experiments.
