The overall goal of the following experiment is to use ultrasound and endo microscopy to illustrate the angiogenic potential of a pool of factors secreted by cancer cells. This is achieved by first implanting a plug to generate a tumor like environment in the mouse in the absence of tumor cells. Next, the blood vessel density and the percent of the functional blood vessels within the plug are assessed.
In the final step, the morphology of the blood vessels is visualized. Ultimately, the balance between the pro and anti-angiogenic factors secreted by the cells and their effects on angiogenesis can be assessed int, vitally, and at various time points by the two complementary imaging modalities. The main advantage of this technique over existing methods like the conventional matrigel plug assay, is that it allows the analysis of a pool of secreted factors by the cells while utilizing complementary intravital imaging modalities.
Demonstrating this procedure will be she ran F Andra PhD students from my laboratory To use ultrasound for evaluating extracellular matrix or ECM mimic gel plug vascularization begin by placing one 1.5 milliliter einor tube containing 80 microliters of freshly prepared conditioned media on ice. The trickiest part of the procedure is injecting the gel without bubbles using ice cold tips. Gentle mixing and a slow injection will ensure an optimal gel administration.
Then using ice cold 1000 microliter tips mix 600 microliters of ice cold four degrees Celsius overnight. Thawed ECM mimic gel into the media taking care not to form bubbles. After confirming sedation by toe pinch, shave the abdomen of each mouse and use an ice cold one milliliter syringe equipped with a 27 gauge needle to slowly subcutaneously.
Inject the contents of one EEND DPH tube per animal. Once the entire volume is administered, hold the needle in place for several minutes until solidification occurs. Three weeks later, shave the abdominal area of the mice again, removing any extra hair with depilatory cream, and then fix the mice in a supine position on the stage of the micro manipulator.
Next, fill a one milliliter syringe equipped with a 30 gauge needle with saline. Then place the needle inside the tail vein. Fix the needle firmly and loosen the syringe.
Now place the transducer in the middle of the desired scanning area and click 3D to initialize the 3D motor stage. Click 3D. Again, input the scan distance and step size and press scan.
Confirm that the entire plug was scanned in the 3D image data displayed. If not, adjust the transducer so that the entire plug is visible and scan the area again as just demonstrated. Next select contrast and adjust the time gain compensation or tgc controls to darken the image, use a vial mixer to activate the microbubbles for 45 seconds.
Then mix 50 microliters of the microbubbles with 50 microliters of saline in a one milliliter syringe. Replace the saline containing loosen syringe with the syringe containing the activated microbubbles and inject the diluted microbubbles into the tail vein of each mouse. Wait a few seconds for the bubbles to reach a steady state and then click 3D and select scan to acquire 3D.
Contrast enhanced images. Click seen a store to save the image data, and then select 3D and destroy 3D. To destruct the microbubbles, click 3D again and select scan to acquire a post destruction cena loop.
Then click seen a store again. Next in the reference settings, click create reference, and then select study management. To open the pre destruction cena loop.
Now click process cena to generate a green contrast overlay. Then select load into 3D in the ultrasound imaging software. Next, click mode settings, click volume and select parallel.
Then segment a series of contours to create a 3D volume of the target tissue and click finish to complete the segmentation. To use fibered confocal endo microscopy to evaluate the ECM mimic gel plug vasculature morphology inject 200 microliters of 10 milligrams per milliliter, fits C labeled dextran into the tail vein of each experimental animal. Then apply a small incision approximately one centimeter from the plug and place the endo microscopy probe gently on top of the plug while keeping the plug intact.
Once the probe is in place, use the foot pedal to press start laser. Once the fluorescence appears on the screen, press select with the foot pedal to acquire short up to 32nd long movies between movies, wash the probe tip in a tube containing water. When the filming is finished, clean the tip with a cotton swab to analyze the mean vessel diameter.
Next, click on detect micro vessels to open the vessel detection module window and click preferences. Make sure that mean vessel diameter in the statistic of interest. Window and display histogram bars in the display mode are marked.
Click detect to display the selected measurements. In order to analyze the fluorescent signal intensity in the areas adjacent to blood vessels, select create circle ROI and create a circle in the area of interest. Finally, select compute histogram within ROI to compute the values related to the circled region.
Here the extensive recruitment of functional blood vessels within U 87 mg conditioned medium loaded plugs is revealed by microbubble contrast enhanced ultrasound imaging. Conversely, within the plugs containing conditioned medium from dormant tumor generating cells, the blood flow is nearly undetectable. The morphology of these newly formed blood vessels is further emphasized by fibered confocal endo microscopy of the vessels after fit c dextran administration as demonstrated in these representative images, note how the blood vessels within the plugs loaded with the U 87 mg conditioned medium exhibit A typical leaky blunt ended enlarged vessel morphology.
The enlarged and highly tangled blood vessels also demonstrate a non-continuous and sluggish flow and leakage of the blood is observed in the surrounding area as well as evidenced by the high fluorescent signal evident outside of the blood vessels. Once mastered, this technique can be done in hours at several time points to optimize the angiogenesis kinetics.
