The overall goal of this procedure is to visualize stem cell endothelial cell interactions in vivo. This is accomplished by first introducing e femoral artery catheter into a sedated animal for intra arterial access. During the second step, the chroma muscle is surgically prepared for intravital fluorescence microscopy.
Next, the stem cell population of interest is injected via the femoral artery catheter. Ultimately, the interactions between the adoptively transferred stem cells and the endothelium of the master muscle microcirculation can be visualized and quantified. This method can help answer key questions in the regenerative medicine field, such as how is the migration of therapeutically applied stem cells regulated within a living organism?
Before beginning the microsurgery D air, a catheter connected to a one milliliter syringe with 0.9%sodium chloride. Then gently shave the anterior aspect of the right scrotum, as well as the right thigh and groin of a 20 to 25 milligram anesthetized male mouse. Collect all the loose hair with a moistened cotton swab, and then place the mouse ventral side up on a plexiglass, custom-made viewing stage consisting of a base plate and a second plate at the coddle edge of the base plate for elevation of both of the hind legs and the scrotum.
After fixing the feet with an elastic drape, place the stage on a heating pad to maintain the animal's body temperature at 37 degrees Celsius. Then using a 10 to 16 fold magnification, use a dissection microscope and a pair of scissors to make an initial incision at the right thigh, just anterior of the femoral vessels from the knee to the groin. Identify the femoral artery and follow it proximally, mobilizing the artery from the surrounding soft tissue.
Then using thermo cautery, identify and cut a large arterial branch leading to the left testicle. Place a six oh prolene stay suture superficially at the left testicle. Then identify and thermo cauterize the large arterial branch running medially at the thigh.
Next, using gentle traction, ligate the femoral artery at the distal part of the right thigh, and then place a suture around the proximal femoral artery as close to the center of the animal as possible. Now, place a loose knot around the femoral artery, but do not tie it. Then apply gentle traction to the suture to create an arterial kinking.
After establishing the kinking, use a pair of micro scissors to carefully size the artery, taking special care not to damage the femoral vein. Next, insert the previously prepared microcatheter into the incision. Carefully loosening the kinking to facilitate passage of the catheter.
Arterial blood flow should be immediately visible within the catheter. Slowly advance the catheter a few millimeters beyond the previous kinking. Then tie down the prepared ligature for fixation of the catheter.
Now gently flush the catheter with the sodium chloride and then aspirate the solution to confirm the correct position of the catheter. Use a piece of tape to further secure the catheter to the operation stage. After the catheter has been adequately fixed and moved aside, cut the skin and fascia above the ventral aspect of the right scrotum.
Taking care not to touch the underlying tissue with any instruments. Extend the incision up to the inguinal fold and coddly to the distal end of the scrotum. Then carefully separate the underlying soft and connective tissues above the cremaster sac without touching the tissue itself.
Then place a six oh prolene stay suture at the distal end of the cremaster to slightly extend the muscle. Next, gently pull the muscle coddly and anteriorly to resect the remaining soft tissue beneath the muscle sac. After the complete separation from the connective tissue, use a pair of scissors to open the C master sac at the cranial edge and extend the incision from this opening down to the distal end of the muscle.
After thermal cauterizing the small vessel connecting the testicle on the cremaster, use scissors to cut the remaining connections between the muscle and the testicle. The opened cremaster will now lay flat on the stage. Now place four six oh prolene Stay sutures at the edges of the tissue and fix them with the elastic drape.
In order to spread the cremaster muscle tissue from this point onward, use a one milliliter syringe to continuously moisten the tissue with PBS. After the preparation has rested for 15 minutes, inject 0.05 to 0.1 milliliter of 1%rodine dextrin via the left femoral artery for contrast enhancement of the micro vasculature. Now, move the mouse and stage under a charge coupled device.
Video camera connected fluorescence microscope, modified for epi illumination, taking special care not to dislocate the femoral artery catheter. After adjusting the water immersion objective for sufficient visualization of the chma microcirculation stochastically defined six post capillary ES for later analysis of firm stem cell adherence. Next load a one milliliter syringe with 200 microliters of the previously labeled fluorescent donor bone marrow stem cells.
Gently tap the syringe to resus, suspend the cell suspension, and then administer the cells through the femoral catheter in 40 microliter injections for a total of five consecutive injections. Record the stem cell rolling immediately after cell injection. Defining rolling as a more than 50%reduction of the cell velocity along the endothelial lining in combination with the typical cellular stick and release movements.
After completion of the intra vital microscopy, carefully remove the cremaster muscle and store it for later analysis. In general, the interaction of directly injected stem or progenitor cells with the vascular endothelium within the cremaster muscle microcirculation is a rare event and occurs exclusively in the post capillary les due to the fluorescent labeling. The rolling cells as indicated by the black arrowhead and the firmly adhering stem cells as indicated by the white arrows, can be clearly distinguished from the circulating endogenous leukocytes in the es.
The micro circulatory conditions represented by the blood flow velocity and wall shear rate usually do not differ significantly between the respective experimental groups with different chemokine treatment. Local treatment of the ker master muscle tissue with different chemokines or mediators of inflammatory response. For examples, stromal cell derived factor one alpha or tumor necrosis factor alpha is capable of mimicking specific microenvironmental conditions.
Such conditions enhance the stem cell endothelial cell interactions to varying degrees depending on the specific chemokine or mediator applied and the stem cell population injected. After watching this video, you should have a good understanding of how to visualize stem cell and athe cell interactions in vivo through intravital microscopy of the CreER muscle microcirculation.
