Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Damage to the adult corneal stroma leads to a rapid wound healing response that compromises one's vision due to opaque scar tissue formation. This protocol generates wounds in embryonic chick corneas which unlike adults, can heal without a scar. Given the intrinsic capacity of the injured embryonic cornea to undergo a complete recapitulation of the native corneal structure with no detectable scarring, the embryonic chick serves an important animal model for elucidating the molecular and cellular factors for scarless corneal wound healing.

The most challenging aspect of this technique is generating a consistent wound that penetrates both the outermost epithelium and the deeper corneal stroma. As one is learning, it may be helpful to view the wounded corneas in a cross section so that the depth of the wound penetration into the corneal stroma can be assessed. To begin, arrange the eggs horizontally on a tray and mark them at the top of the egg to denote the expected position of the embryo.

Incubate these eggs in a 38 degree Celsius humidified incubator with the rocking function activated. Remove the egg from the incubator on the third day of embryonic development and position it horizontally in a secure egg holder. Create a small hole in the top of the egg shell near the pointed end of the egg using the sharp end of dissecting scissors.

Insert an 18 gauge beveled hypodermic needle through the hole. With the needle pushed to the bottom of the inner surface of the egg and the bevel side of the needle facing the pointed end of the egg, remove two to three milliliters of the albumin from the chicken egg and discard. Clean the eggshell surface surrounding the hole with lint free wipes, lightly dampened with 70%ethanol and wipe dry.

Seal the hole made for removing albumin with clear tape. With the sharp end of dissecting scissors, make a second window hole in the top of the eggshell. Ensure that the scissors do not extend too far into the egg shell to avoid contacting and damaging the embryo or embryonic vasculature positioned directly below the side of the second hole.

Using curved forceps, widen the window hole to span approximately two to three centimeters in diameter to reveal and gain access to the developing embryo beneath the shell. Now, insert one end of the forceps into the hole, keeping it parallel to and closely juxtaposed with the eggshell. With the other forceps end positioned outside the eggshell, carefully pinch the two forceps ends together, allowing them to break and remove small pieces of the eggshell.

Continue breaking and removing eggshell fragments until there remains a two to three centimeter window that directly overlays the embryo. To limit bacterial contamination, add 100 to 200 microliters of ringer solution containing 50 units per milliliter of penicillin and 50 micrograms per milliliter of streptomycin through the window hole. Seal the window hole using clear adhesive tape.

Perform this egg sealing by aligning a corner of the tape on the long axis of the hole and pressing the tape to the shell one to two centimeters away from the edge of the hole. Continue sealing around the opening until a hanging flap of tape is left on one side. Press the two pieces of tape together, creating a domed shape over the hole and press the flap of overhanging tape to the shell to finish sealing the egg.

Return the windowed eggs to the incubator for further development. Ensure to keep these eggs horizontal and turn off the incubators rocking function. Use a dissecting microscope to ensure the embryo is at the proper developmental stage and locate the positions of the amniochorionic membrane and the chorioallantoic membrane.

Then use sterilized micro dissecting scissors to cut a hole in the amniochorionic membrane directly above the forelimb that extends from the membrane, overlying the forelimb to the membrane overlying the head. Use two pairs of fine sterile forceps and gently grab the amnion in two adjacent positions between the amniochorionic membrane ACM and the chorioallantoic membrane. Carefully move each pair of forceps, both firmly gripping the amniotic membrane, away from one another with one pair moving dorsally to the embryo and the other ventrally.

Use sterilized fine forceps to dissect and remove any remaining amnion membrane covering the embryo. Using sterilized forceps, grasp the amnion near the mid cranial region of the embryo and carefully pull the amnion in a coddle direction with respect to the embryo toward the earlier displaced chorioallantoic membrane. Reseal the window hole using clear tape as described in the manuscript and return the egg to the incubator for further development.

The extra embryonic membranes may need to be repositioned to assure the right eye is accessible. If the right eye is not accessible for wounding due to the position of the embryo in the egg, it can serve as a non wounded control. Use a micro dissecting knife to make an incision that spans the extent of the cornea of the right eye which is parallel to and inline with the choroid tissue.

Use the micro dissecting knife to again, lacerate the cornea in the same spot as the first incision, two times more in such a way that the cut two and cut three occurring along with the same position in the cornea as cut one. To help with viability, use the curved iris forceps to tuck the embryo back under the chorioallantoic membrane after surgery to promote proper growth of the chorioallantoic membrane. Next, add three to four drops of ringer solution containing antibiotics to hydrate the embryo and sterilize the egg.

Reseal the window hole with clear tape and return to the incubator, leaving the egg horizontal. Allow the embryo to develop and the corneal wound to heal for a desired period. After the incubation, euthanize the embryo and use curved iris forceps to harvest the eye in a Petri dish of Ringer's saline solution by gently grasping the eye on its posterior side where the eye and facial tissue meet and carefully lifting the whole eye away and free from the facial tissue.

Use fine forceps to poke a small hole of three to five centimeters in the back of the whole eye and fix the whole eye in 4%paraformaldehyde at four degrees Celsius overnight with mild agitation. To achieve an ideal wound, it is crucial to use a sharp micro dissection knife and apply the correct amount of pressure during the laceration. Applying too little pressure will result in a shallow wound that tears the corneal epithelium without sufficiently penetrating the anterior stroma.

Applying too much pressure results in a full extent wound penetrating the entire stroma and exposing the aqueous humor to the external environment. Carrying out the proper lacerating incisions produces an ideal wound that initially enlarges zero to three days post wounding. After that, re-epithelialization and new tissue formation occurs to ultimately close the wound in a scar free fashion by 11 days, post wounding.

Spatiotemporal localization of the extracellular matrix proteins, fibronectin and tenascin within the healing wound was found to be elevated at time points corresponding to corneal re-epithelialization, suggesting their involvement in wound closure, epithelial cell migration and survival. Whole mount immunohistochemistry revealed that the nerves that directly juxtaposed the wounded central cornea are temporarily inhibited from the healing corneal tissue. Despite earlier inhibition, corneal nerves eventually innervate the fully healed corneal tissue 11 days post wounding to similar density levels and in similar patterns to stage matched non-wounded controls embryonic day 18.

As evidenced by second generation harmonic imaging, bundles of collagen fibers throughout varying depths of the central cornea wound are seen arranged orthogonally, matching the native macro structure of non wounded central corneal tissue. When combined with classic developmental biology approaches, such as tissue grafting and bead implantation, as well as modern day approaches for gene manipulation such as retroviral infection and DNA electroporation, this animal model promises to reveal molecular factors and cellular mechanisms necessary to promote scarless cornea wound healing. Determining key molecular factors in matrix proteins that regulate fetal scarless wound healing will pave the way for therapies that foster a more restorative healing process with less scarring and better recapitulation of the normal tissue architecture.