This protocol allows the visualization of epithelial cells during wound healing in live animals. It allows us to see how cells behave in their natural complex environment. This technique introduces reagents into the extracellular matrix and the cell cytoplasm to study the effect of these reagents on cell spreading and migration during wound healing in live animals.
Epithelial wound healing and Clytia remarkably resembles healing in more complex animals. Therefore, we can learn a lot from this system about the mechanisms of wound healing and how they originated. Demonstrating these procedures will be Elizabeth Lee, and research technician Emily Watto.
To begin the process for wounding, prepare a modified transfer pipette by cutting the pipette tip with scissors, creating an enlarged opening measuring about 0.5 to 0.7 centimeters in diameter. Using the modified transfer pipette, place the medusa collected from the established polyp colonies on a depression slide, with the medusa X umbrella facing upward. Ensure that just enough of artificial seawater, or ASW, covers the animal.
To create micro wounds within and between cells, place a cover slip over the animal. The cover slip compresses the mesoglea and the rebound of the compressed tissue forces the cells slightly apart. Image the animal immediately to observe the created micro wounds.
To create small epithelial wounds, place the medusa on a depression slide with the medusa X umbrella facing up, using a modified transfer pipette, as demonstrated earlier. Using a 200 microliter pipette tip, gently scratch the surface of the medusa. Gentle scratching may create rips in the basement membrane.
Cover the animal with a cover slip for imaging. Placing the cover slip is sometimes sufficient to create small epithelial wounds even without scratching. Prepare a microinjection needle, using a micro pipette pillar and glass capillary tube, following the steps outlined in the text.
Place the empty microinjection needle into a microinjection holder affixed to a micro manipulator and cut the tip of the needle, such that the opening is approximately 20 to 40 microns. Set the hold pressure on the microinjector to zero, and the ejection pressure to approximately 20 pounds per square inch. Set the microinjector to deliver a two second pulse of air.
To create large epithelial wounds, place the medusa on a depression slide, as demonstrated before. Using the micro manipulator, adjust the microinjection needle tip to remain just above the water, by carefully dipping the tip into the water, and then retracting it such that it is close to the medusa's epithelial surface. Pulse air by pressing start on the injector.
Repeat the pulse in the same spot two to four times, depending on the width of the tip. Larger tips require fewer pulses. Cover the wounded animal with a cover slip for imaging large wounds.
Adjust the focus to the x umbrella. Hexagonal cells should be clear. Manually identify a wound to image it.
Start a program that collects images as a movie in real time, or one that collects a series of images at regular intervals. Monitor the progress to ensure that the wound area does not drift out of the field of view, and that the cells of interest remain in focus. For injecting dyes and drugs, make a microinjection needle following the steps outlined in the text.
Backfill the microinjection needle using a long pipette tip with an excess volume of dye or drug for injection into the medusa. Using a modified transfer pipette, place a medusa with the sub umbrella facing up into the polydimethylsiloxane, or PDMS injection dish, with just enough ASW to cover the animal. Place the dish on a stage of a dissecting microscope.
Focus on the microinjection needle tip, and advance it into the water near the medusa. With the micro manipulator, press the needle into the dish until it bends and breaks. Creating a tip opening of approximately 10 to 20 microns.
Using the micro manipulator, insert the tip of the needle through the sub umbrella into the mesoglea without puncturing the X umbrella. On the microinjector, set the hold pressure to zero, and the injection pressure below or equal to 20 pounds per square inch. Inject into one or two quadrants, filling each with a spot of dye or drug roughly one fourth of the area of that quadrant.
Depending on what dye or drug is being injected, animals are placed into a beaker of fresh ASW to allow for dye or drug diffusion and incubation. For imaging, mount the medusa onto a depression slide using a modified transfer pipette, positioning the animal with the X umbrella facing up. Animals can be wounded at this stage to test the effect of an injected reagent.
The micro wounds within and between cells formed small lamellipodia during wound closure. This was followed by contraction, and the wounds healed in less than a minute. The small epithelial wounds also healed through lamellipodia formation and extension of lamellipodia contacts.
Rapid and progressive wound closure preceded tissue contraction along the newly formed wound seam. The normalized healing rate of two small wounds expressed as the percentage of the total wound area over time indicated some variability in wound closure dynamics. Data from 14 different wounds were used to establish an average wound healing curve in untreated animals.
In a small wound having basement membrane damage, the marginal cells spread around the damaged area, and the gap closed with a purse string contraction. When the tissue was dehydrated or too damaged to repair, cell movements could stop, or the entire sheet of cells could burst. Large wounds healed in several stages.
Smoothing of the edge following contractions at the margin, lamellipodia formation, and extension of lamellipodia contacts collective cell migration at, and several cell tiers away from, the margin closed large gaps. Nuclear and membrane staining were achieved by microinjecting the reagents into the ECM. Microinjection of cytochalasin B inhibited lamellipodia formation post-wounding.
Handle the animals gently. When injecting reagents, make sure the tip of the microinjection needle is in the mesoglea, and has not gone all the way through the animal. Currently, transgenic animals are being made with fluorescent tag proteins, combined fluorescence and differential interfering contrast microscopy will give an even clearer image of the cellular events in epithelial wound healing.
