The overall goal of this procedure is to isolate endothelial cell tubes from the mouse superior epigastric artery or SEA to study the intra and intercellular signaling dynamics of a native intact microvascular endothelium. This is accomplished by first isolating the SEA from the mouse abdominal skeletal muscle wall. In the second step, the vessel is gently and somatically digested, and then carefully TATed to dissociate the smooth muscle cells and adventitia from the endothelial cell tube.
In the final step, the tube is secured in a flow chamber and super fused with a physiological saline solution. Ultimately, confocal imaging can be used to visualize calcium signaling in the native intact microvascular endothelial tube. The main advantage of this technique over existing methods like culturing endothelial cells, is that the endothelial cells that comprise the tube and maintain their native morphology, protein expression, and responsiveness, The endothelium is integral to blood flow control throughout the body.
The application of this technique allows us to investigate the cell to cell signaling events that mediate blood flow control and how they may go awry during vascular dysfunction. To isolate the SEA first, make a small incision through the skin just above the area of the pubic region of an anesthetized mouse, extend the incision laterally in each direction to the respective hind limbs, and then continue the incision roly along the ventral midline to the top of the rib cage, further extending the incision laterally in each direction to the respective four limbs. Now gently lift the skin and sever the connective tissue holding the skin to the underlying muscle, exposing the entire surface of the abdominal musculature.
Irrigate the exposed muscle with room temperature saline solution. Then under a stereo microscope, lift the fat pad located at the bottom of the sternum. Make an incision through the fat pad and along the bottom rib.
The SEA should now be visible, taking care not to damage the artery, irrigate the exposed tissue with more room temperature, saline solution. After the top layer of skeletal muscle has been retracted, note the length of the SEA and then carefully excise the thin muscle layer underneath the artery. Next, use angled forceps to pass a length of six oh silk suture underneath the SCA.
Then ligate the artery and its adjacent vein to keep it pressurized and to keep the blood retained within the vessel lumen. After ligating the SCA on the contralateral side, make an incision along the midline of the abdominal muscles to separate the respective sides, and then extend the incision laterally in each direction as done for the skin. Continue the incision vertically along the outer edge to completely separate the abdominal muscle from the body.
Then cut the SEA above the ligation to maintain the seal and place the isolated muscle and artery in a 50 milliliter beaker containing 10 milliliters of four degrees Celsius dissection buffer. After isolating the muscle from the other side of the abdomen, incubate the tissues in dissection buffer for 10 minutes. Now, place the abdominal muscle containing the SEA in a four degree Celsius Petri dish coated with a layer of cyl guard and containing dissection buffer Use 0.15 millimeter insect pins to stretch the SEA and muscle to their previously noted approximate in vivo lengths.
Secure the SEA to the cyl guard orienting the muscle such that the thin layer facing the peritoneum is on top. Then working from the upstream site of ligation towards the downstream end clear about one to two centimeters of the SEA from its paired vein and the surrounding tissue until the first major branch site. Cut the SEA just above the branch site and just below the ligation.
Next, use a piece of elastic tubing to attach the backend of a pipette to a five milliliter syringe containing ice cold dissection buffer. Secure the cannulation pipette tip within the dissection chamber and cannulate the SEA. Then once all of the erythrocytes have been flushed out, remove the SEA from the cannulation pipette and remove the pipette from the dissection dish to isolate the endothelial tubes.
First, fill a 12 by 75 millimeter glass culture tube halfway with ice cold dissection buffer. Then after cutting the SEA into one to three millimeter pieces, use angled forceps to transfer the arterial pieces into the culture tube and place the tube on ice. Next, combine digestion enzymes with the dissociation buffer to a final volume of one milliliter in a separate 12 by 75 millimeter culture tube and preheat the enzyme solution to 37 degrees Celsius with a heating block.
Remove the culture tube containing the arterial pieces from four degrees Celsius and place it at room temperature to warm up while the enzyme solution is warming to 37 degrees Celsius. Carefully aspirate the now room temperature dissection buffer from the culture tube, leaving a small volume containing the vessel segments. Now slowly add room temperature, dissociation buffer without enzymes to the vessel segments such that the arterial pieces remain on the bottom of the culture tube to wash away any remaining dissection buffer.
Once the enzyme solution has reached 37 degrees Celsius, aspirate the dissociation buffer from the culture tube containing the vessel segments again, leaving a small volume containing the vessel segments. Now transfer the 37 degrees enzyme solution to the culture tube and incubate the culture tube in the heating block for 30 minutes at 37 degrees Celsius. During the incubation, prepare a literation pipette backfilled with mineral oil, score the pipette, make a clean break, break the pipette with forceps fire, polish the tip of the pipette and secure it on a micro syringe mounted in a micro manipulator.
Then retract the micro syringe plunger to fill the pipette with two nanoliters of dissociation buffer and position the pipette tip over the flow chamber at the end of the incubation. After carefully aspirating the buffer as just demonstrated, wash the arterial segments with four milliliters of room temperature dissociation buffer. Next, gently aspirate one vessel segment with a one milliliter micro pipette and place the vessel into a flow chamber with one milliliter of the dissociation buffer.
Position the tip of the tation pipette near one end of the vessel segment, and then aspirate the vessel segment into the tating pipette at about 225 nanoliters per second, so as not to cause mechanical strain to the endothelial cells, eject the vessel back into the chamber. If the digestion was successful, the smooth muscle cells and adventitia will be dissociated from the endothelial tube. Move the adventitia away from the endothelial tube as soon as possible to keep it from entangling the tube, and then repeat the tation as just demonstrated until all the smooth muscle cells are dissociated.
After the final iteration, ASEE and placed the isolated endothelial tube in the center of the flow chamber, aligned along the direction of flow and removed the triggering pipette. Secure pinning pipettes in micro manipulators mounted at either end of the flow chamber and then position their tips at the respective ends of the endothelial tube. Lower the pinning pipettes, one at a time over opposite ends of the tube, pressing the tube against the bottom of the chamber, about 50 micrometers from each end of the tube.
Just as the pinning pipette touches the tube, slowly retract it along the axis of the tube, extending it to its approximate in vivo length. Press the pipette against the chamber bottom to secure the tube, and then begin the flow of the superfusion solution using a peristaltic pump to maintain a constant flow of the superfusion solution across the endothelial tube. In this differential interference contrast image, an isolated endothelial cell tube from an SEA following, a one hour superfusion with superfusion buffer at three milliliters per minute at room temperature is shown.
This movie demonstrates the calcium responses of a flu oh four loaded endothelial cell tube in response to a one micromolar acetylcholine stimulation. These representative fluorescence images were collected at various time points after endothelial tube stimulation with acetylcholine. Note how the intracellular calcium oscillates over time.
While attempting this procedure, it's important to remember to avoid mechanically damaging the endothelial tube during tri positioning and pinning as the endothelial cells are extremely delicate and easily damaged. After watching this video, you should have a good idea of how to isolate endothelial tubes from the mouse superior epigastric artery, and thereby study native intact microvascular endothelium.
