Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Summary

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. 

Abstract

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

Introduction

The cornea is the transparent, outer-most tissue of the eye that transmits and refracts light conducive to visual acuity. In the adult cornea, damage or infection to the corneal stroma leads to a rapid and robust wound healing response characterized by keratocyte proliferation, fibrosis, increased inflammation leading to cytokine-induced apoptosis, generation of repair myofibroblasts, and overall remodeling of the extracellular matrix (ECM)^1,2. Following injury, such corneal tissue repair results in opaque scar tissue that reduces corneal transparency and occludes the passage of light, thus distorting vision and, in the most severe cases, leading to corneal blindness³. Thus, there is a clear need to develop reliable animal models to address the complexities of wound healing and to identify the cellular and molecular factors responsible for wound closure and tissue regeneration.

To date, most studies examining corneal wound healing have utilized post-natal⁴ or adult animal models^1,2,5,6,7. While these studies have led to a significant advancement in the understanding of the corneal wound healing response and the mechanisms underlying scar formation, the damaged corneal tissues in these healing models fail to fully regenerate, thus limiting their utility for identifying the molecular factors and cellular mechanisms responsible for fully recapitulating corneal morphology and structure post-injury. By contrast, fetal wounds generated with a knife in the embryonic chick cornea possess an intrinsic capacity to heal fully in a scarless fashion⁸. Specifically, the embryonic chick cornea exhibits nonfibrotic regeneration with the complete recapitulation of the extracellular matrix structure and innervation patterns^8,9.

The present protocol describes a sequence of steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to facilitate access to the embryo. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed and wounding is desired. Third, linear central cornea incisions penetrating through the corneal epithelium and into the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.

Protocol

The strain of eggs used in this protocol was White Leghorn, and all animal procedures were approved by the Institutional Animal Care and Use Committee at Illinois Wesleyan University.

1. Incubation of chick eggs

1. Keep the eggs at ~10 °C for up to 1 week after they are laid to halt development. When ready to initiate chick embryo development, wipe the entire eggshell with lint-free wipes (see Table of Materials) saturated with room temperature water to remove dirt and debris.
2. Ensure the eggshell is sanitized. Wipe down the entire egg surface with lint-free wipes dampened with 70% ethanol. Quickly wipe off the ethanol to dry the egg and avoid ethanol absorption through the eggshell to the embryo.
3. Arrange the eggs horizontally on a tray. Mark the top of the egg to denote the expected position of the embryo. Incubate the eggs horizontally, with the rocking function activated, in a 38 °C humidified incubator.

2. Windowing the eggs to prepare for membrane dissection

1. Remove eggs from the incubator on the third day of embryonic development (E3). Sterilize the top of the eggs with lint-free wipes dampened with 70% ethanol. Dry the ethanol from the eggshell surfaces.
   NOTE: To ensure that development was not delayed during the windowing procedure, 6-12 eggs were removed, and the procedure was quickly carried out on these while the remaining eggs were left in the incubator.
2. Position an egg horizontally in a secure egg holder (see Table of Materials). Using the sharp end of dissecting scissors, create a small hole in the top of the eggshell near the pointed end of the egg.
   NOTE: This hole will facilitate the removal of albumen, which is necessary to drop the yolk and embryo away from the inner eggshell surface. For egg holders, paper-pulp egg filler flats, within which the eggs come shipped, were used.
3. Through the hole (Step 2.2.), insert an 18 G beveled hypodermic needle. With the needle pushed to the bottom inner surface of the egg and the bevel-side of the needle facing the pointed end of the egg (e.g., away from the expected location of the yolk and the embryo near the middle of the egg), remove 2-3 mL of albumen from the chicken egg and discard.
   NOTE: If one's needle nicks the embryo or its associated vasculature during this step, it will result in blood being aspirated with the albumen. This will result in embryo death. Further, if the yolk is inadvertently aspirated along with the albumen during this step, the embryo will not be viable. In either case, the egg should be discarded if the embryo cannot be immediately used for other purposes.
4. 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 albumen with clear tape.
5. With the sharp end of dissecting scissors, make a second "window" hole in the top of the eggshell at the marking site (Step 1.3.). Ensure that the scissors do not extend too far into the eggshell to avoid contacting and damaging the embryo or embryonic vasculature, which will often be positioned within the egg directly below the site of the second hole.
6. Using curved iris forceps, widen the "window" hole to span ~2-3 cm in diameter and serve as a "window" to the developing embryo beneath the shell.
   1. 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 2-3 cm window that directly overlays the embryo.
      NOTE: Eggs in which embryos do not lay directly beneath the hole made in Step 2.6. must not be used as the forthcoming membrane dissections would be challenging to complete. Even with rocking the eggs horizontally, about 10% of the eggs are unusable due to the poor positioning of the embryo. These embryos can be used for other purposes.
7. To limit bacterial contamination, add through the window hole (e.g., into the egg) ~100-200 µL of Ringer's solution (8 g of NaCl, 0.37 g of KCl, and 0.23 g of CaCl₂.2H₂0 per L of distilled H₂0) containing Penicillin/Streptomycin antibiotics (50 U/mL of Penicillin and 50 µg/mL of Streptomycin, see Table of Materials).
8. Seal the window hole using clear adhesive tape. Perform the egg sealing by aligning a corner of the tape on the long axis of the hole and pressing the tape to the shell ~1-2 cm away from the edge of the hole.
   1. 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 over-hanging tape to the shell to finish sealing the egg.
      NOTE: Eggs need to be windowed at either E2 or E3. As per experience, windowing prior to E2 results in low embryo viability. Moreover, by E4, the embryo and extraembryonic membranes become attached to the eggshell¹⁰, and any attempts to window at E4 or later often result in embryo damage or tearing of extraembryonic blood vessels, with either event leading to the outcome of embryo death.
9. Return the "windowed" eggs to the incubator for further development. Ensure to keep the eggs horizontal and turn off the incubator's rocking function.
10. Repeat Steps 2.2.-2.9. for each egg.

3. Microdissections of the extraembryonic membranes

1. Remove an E5.5 windowed egg from the incubator. Expose the embryo by cutting the tape away from the window with sterilized dissecting scissors.
2. Use a dissecting microscope to observe the embryo and its extraembryonic membranes through the window. If necessary, use scissors or curved iris forceps to widen the window so that the embryo is well-positioned beneath the window, taking care not to damage the embryonic vasculature.
   1. Add two drops of Ringer's solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.
3. Use a dissecting microscope to ensure the embryo is at the proper developmental stage (Hamburger Hamilton stage 27, ~E5.5)^11,12 and locate the positions of the amniochorionic membrane (ACM) and the chorioallantoic membrane (CAM).
   NOTE: At this stage, the embryo is surrounded by the ACM, which comprises the amniotic membrane and overlying chorionic membrane fused and partially covered by the highly-vascularized allantois, which extends from the embryo's gut region and fuses with the overlaying chorion to form the CAM^11,12. The ACM is not heavily vascularized, which enables one to dissect these membranes to expose the embryo without damaging blood vessels and harming the embryo.
4. Perform extraembryonic membrane dissections at this stage (E5.5).
   NOTE: E5.5 is the ideal time to carry out the extraembryonic membrane dissections. Dissecting the membranes earlier (e.g., at E4) before CAM formation reduces embryo accessibility at later stages¹¹. Moreover, at E5.5, the embryo is only partially covered by the highly-vascularized CAM, yet over the next 1-2 days, the CAM quickly envelops and precludes further access to the embryo^11,12. For this reason, membrane dissection at E6 or later is challenging as the risk of tearing blood vessels increases.
   1. Use a pair of sterilized fine forceps to gently grasp the ACM and pull it away from the embryo. Then use sterilized micro-dissecting scissors to cut a hole in the ACM directly above the forelimb that extends from the membrane overlying the forelimb to the membrane overlying the head.
      NOTE: This step relaxes the chorion and amnion membranes, thus making them easier to grab and further dissect with forceps in the next steps. See Figure 1 for a helpful schematic of how membranes are dissected through the eggshell window.
5. Use two pairs of fine, sterile forceps to gently grab the amnion in two adjacent positions between the ACM and the CAM (e.g., an area between the cut made above the forelimb and the nearest edge of the CAM).
   1. 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.
      NOTE: This motion serves to tear the amnion further while also separating the ACM (which gets pulled in the dorsal direction with respect to the embryo by one pair of forceps) and the CAM (which gets pulled in the ventral direction with respect to the embryo by the other pair of forceps).
   2. Ensure that the membranes are separated when the CAM no longer covers the embryo and the allantoic artery and vein, which emanate from the embryonic gut to the CAM, are readily apparent.
6. Use sterilized fine forceps to dissect and remove any remaining amnion membrane covering the embryo. It is most commonly observed that the remaining amnion will partially cover the caudal half of the embryo.
   1. Using sterilized forceps, grasp the amnion near the mid-cranial region of the embryo and carefully pull the amnion in a caudal direction with respect to the embryo toward the earlier displaced CAM. The embryo will now be fully exposed, and further growth of the CAM will mainly occur away from the developing embryo.
      NOTE: See Figure 1 for a helpful schematic on how the exposed embryo will appear following membrane dissection. Also, refer to a previously published report¹¹ for helpful schematic diagrams of Steps 3.3.-3.6.
7. Add a few drops of Ringer's solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.
8. Reseal the window hole using clear tape, as described in Step 2.8. Return the egg to the incubator for further development, keeping the egg horizontal and keeping the incubator's rocking function inactivated.
9. Repeat Steps 3.1.-3.8. for each egg.
   NOTE: The extraembryonic membrane dissections at E5.5 described above will enable access to the embryo through E7, which is when wounding can be carried out^8,9. By E8, the continued growth of the CAM tissue begins to cover the cranial region of the embryo, thus precluding further access to the cornea. If one desires to carry out wounding in older E8-E9 corneas, it is possible to reposition the growing CAM ventrally with respect to the embryo (Step 3.10.). If one wishes to wound at E7, Step 3.10. is not necessary to perform and one may proceed to Step 4, corneal wounding.
10. Remove an E7 egg from the incubator whose extraembryonic membranes were previously dissected at E5.5 (Steps 3.1.-3.8.). Grasp with sterilized forceps any available amnion membrane tissues fused to the CAM and gently pull the amnion membrane away from the cranial region of the embryo in a ventral direction with respect to the embryo.
    ​NOTE: Since the amnionic membrane being displaced away from the embryo is fused to the CAM, the growing and highly vascularized CAM will follow the amnion-grasping forceps and move away from the cranial region. Repeat this step daily to continually displace the CAM away from the embryo until the embryo is at the desired age for wounding.

4. Corneal wounding

1. Obtain an egg from the incubator for wounding at the desired embryonic age, E7-E9. Expose the embryo by cutting the tape away from the window with sterilized dissecting scissors. Add a few drops of Ringer's solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.
2. Use a micro-dissecting knife to make an incision that spans the extent of the cornea of the right eye (due to how the embryo lays in the egg, the left eye is not accessible but can serve as a non-wounded control), which is parallel to and in line with the choroid fissure (Figure 1). The first cut will traverse the corneal epithelium.
   1. Use the micro-dissecting knife to again lacerate the cornea in the same spot as the first incision 2x more (e.g., three cuts total, with cut 2 and cut 3 occurring along with the same position in the cornea as cut 1)¹¹. The second laceration will traverse the basement membrane, and the third will penetrate the anterior stroma.
      NOTE: If the embryo has settled under the dissected CAM, one can use sterilized curved iris forceps to carefully move the head out from under the CAM. Position the curved iris forceps beneath the head, making contact with the left side of the head. Cradle the entire head on top of closed curved iris forceps and gently raise the head around and above the CAM. To help with viability, use a similar technique with the curved iris forceps to tuck the embryo back under the CAM after surgery to promote proper growth of the CAM.
3. Add 3-4 drops of Ringer's solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.
4. 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 (e.g., 0.5-11 days), and then humanely euthanize the embryo by decapitation.
5. Use curved iris forceps to harvest the eye from a euthanized embryo floating 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.
   1. Use fine forceps to poke a small hole (3-5 cm) in the back of the whole eye and fix the whole eye in 4% paraformaldehyde at 4 °C overnight with mild agitation.

Results

Following the earlier dissection of the ACM and CAM at E5.5 to expose the cranial region of the developing embryo, a series of lacerations that spanned the E7 central cornea was made in ovo (Figure 1). An ideal wound to study cornea regeneration occurs following three lacerations, each made in the same location of the cornea. The first laceration traverses the corneal epithelium, while the second and third lacerations penetrate the underlying basement membrane and anterior stroma, r...

Discussion

The chick is an ideal model system for studying fetal, scarless cornea wound repair. Unlike mammals, the chick is easily accessible throughout development using in ovo⁸ or ex ovo strategies²⁴. The embryonic chick cornea is much larger than rodent corneas, with nearly 50% of the cranial volume dedicated to the eye²⁵, making it highly amenable to physical manipulations such as wounding. Moreover, chicken eggs are readily availabl...

Materials

Name	Company	Catalog Number	Comments
18 G hypodermic needle	Fisher Scientific	14-826-5D
30 degree angled microdissecting knife	Fine Science Tools	10056-12
4′,6-diamidino-2-phenylindole (DAPI)	Molecular Probes	D1306
5 mL syringe	Fisher Scientific	14-829-45
Alexa Fluor labelled secondary antibodies	Molecular Probes
Calcium chloride dihydrate (CaCl2-H20)	Sigma	C8106
Chicken egg trays	GQF	O246
Dissecting Forceps, Fine Tip, Serrated	VWR	82027-408
Dissecting scissors, sharp tip	VWR	82027-578
Iris 1 x 2 Teeth Tissue Forceps, Full Curved	VWR	100494-908
Kimwipes	Sigma	Z188956
Microdissecting Scissors	VWR	470315-228
Mouse anti-fibronectin (IgG1)	Developmental Studies Hybridoma Bank	B3/D6
Mouse anti-laminin (IgG1)	Developmental Studies Hybridoma Bank	3H11
Mouse antineuron-specific β-tubulin (Tuj1, IgG2a)	Biolegend	801213
Mouse anti-tenascin (IgG1)	Developmental Studies Hybridoma Bank	M1-B4
Paraformaldehyde	Sigma	158127
Penicillin/Streptomycin	Sigma	P4333
Potassium chloride (KCl)	Sigma	P5405
Sodium chloride (NaCl)	Fisher Scientific	BP358
Sportsman 1502 egg incubator	GQF	1502
Tear by hand packaging (1.88 inch width)	Scotch	n/a