Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

Hello, my name is Christian Deffge. I'm a medical doctor at university hospital in Magdeburg and I'm working in the cardiovascular research group of Dr.Herold and Professor Braun-Dullaeus. We investigate the augmentation of collateral growth.

Therefore, we use imaging. In this video, we want to explain our methods. Resuspend the cells in serum-free culture medium with a density of one times 10 to the sixth cells per milliliter.

Add five microliters of one millimolar DIO and dimethylformamide to one milliliter of the cell suspension and resuspend carefully. Incubate the cell solution at 37 degrees Centigrade for 20 minutes. Centrifugate the cells at 500 g for five minutes.

Dislodge the supernatant and resuspend the cells with 37 degrees Centigrade warm FCS supplemented medium. Repeat this step twice. Count the cells with the help of a Neubauer counting chamber and light microscopy.

Resuspend the cells with sodium chloride solution. Inject the cells into the tail vein. Matrigel matrix is a reconstituted basement membrane preparation extracted from the squamous sarcoma.

It screens test molecules for either endothelial cell network formation or anti-cancer therapies through angiogenic inhibition. Reassess the tumor angiogenic potential of monocytes we strive to use for cell therapy. Add 100 nanogram BFGF, 300 nanogram VEGFA and 26 international units heparin under sterile conditions to the Matrigel.

After vortexing the solution, fill it up into one milliliter insulin syringe and store on ice until usage. For better visibility of the subcutaneous Matrigel plaque, shave the skin of the mouse at the injection site. You can use a sharp scalpel or depilatory cream.

Lay the animal on the table and hold the skin of the mouse next to the injection site on the flank. Inject 500 microliters of Matrigel subcutaneously. For practical reasons, it is necessary to inject the Matrigel compactly at one location to avoid subcutaneous dispersion.

It will be easier to dislodge the Matrigel matrix from the tissue after sacrificing the mouse at the end of the experiment. Practice the monocyte injection with sodium chloride solution before experimentation. If the monocytes cannot be applied systemically, there will be no systemic effect.

Within this protocol, we injected 2.5 million monocytes. Try to inject no more than five microliters per gram. Make sure the mouse is warmed with the help of a heating pad while the whole procedure.

For systemic application of the cells, you will need a restrainer and one milliliter insulin syringe, a 30 g needle and the monocytes resuspended and sodium chloride solution. Carefully handle the mouse and restrain the animal in the restrainer. Make sure the mouse is not harmed and has adequate space for breathing.

Put the restrainer on the heating pad so that the tail can contact the plate. Make sure you disinfect the injection site to avoid an infection. Identify the tail veins which are located on the lateral side of the tail.

Turn the tail 90 degrees so the tail vein appears on the upper side of the tail. Try to inject the monocyte solution in a flat angle. Observe the animal for 30 minutes to look for systemic side effects.

Place the mouse in its cage after the animal has fully recovered. Place the anesthetized mouse on the prewarmed microscope stage after the microscope has reached stable conditions and the settings have been tested by a dummy. Fix the paws with adhesive tape.

Disinfect the skin at the site of the leg or flank that is used for microscopy. Shave the region of interest for better handling and to avoid interference with hair. Keep the region of interest moist.

Otherwise, the quality of images and tissue will be compromised. Place the leg between two adjustable stems. Position a cover glass on top of the stems.

Start the microscopy and adjust the position of the leg if needed. Microscope settings depend on the software that is used. Please see the protocol for further information.

Our data demonstrates that intravital microscopy represents a potent tool for visualization of transplanted monocytes within the blood flow in small animal models. The monocytes must be injected into the venous system to maintain systemic effects and to avoid emboli which can occur if the injection is conducted in the arterial system. If Matrigel is used, injecting slowly to avoid dispersion will help with plaque explantation for further histological examination.

Within the Matrigel plaque, we are able to measure vascularization by counting capillaries within different experimental settings. Intravital microscopy findings can be verified by histological examination. We are able to find monocytes detected by intravital microscopy within all tissue sections.

If much time is required for the acquisition of images, the amount of rhodamine dextran within the vessel decreases. For better visibility of the vessels, the application of the rhodamine dextran must be repeated. The use of positive probes for cells on Matrigel can help obtain optimal settings.

The goal for peripheral arterial disease or ischemic heart disease therapeutics is to increase blood flow to other perfused areas caused by hemodynamic stenosis. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is a noninvasive option for patients without indications for surgery. In addition to the development of collateral arteries in ischemic areas, monocytes can also influence the growth of tumors.

To investigate these processes, we use Matrigel injection in connection with intravital microscopy. Our aim with this protocol is to demonstrate an easy and efficient way to study immunological processes caused by ischemia in an in vivo model. With this new method, we are able to generate a more realistic test environment compared to histological workup of postmortem muscle tissue.