This work was supported by an Artistic and Scholarly Development grant through Illinois Wesleyan University to TS and funded in part by NIH-R01EY022158 (PL).

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
<ol>
	<li>Keep the eggs at ~10 &#176;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 w...

<ol>
	<li>Wilson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+wound+healing.">Corneal wound healing.</a> Experimental Eye Research. 197, 108089 (2020).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in Retinal and Eye Research. 49, 17-45 (2015).</li><li>Whitcher, J. P., Srinivasan, M., Upadhyay, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+blindness:+a+global+perspective.">Corneal blindness: a global perspective.</a> Bulletin of the World Health Organization. 79 (3), 214-221 (2001).</li><li>Ritchey, E. R., Code, K., Zelinka, C. P., Scott, M. A., Fischer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+cornea+as+a+model+of+wound+healing+and+neuronal+re-innervation.">The chicken cornea as a model of wound healing and neuronal re-innervation.</a> Molecular Vision. 17, 2440-2454 (2001).</li><li>Berdahl, J. P., Johnson, C. S., Proia, A. D., Grinstaff, M. W., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+sutures+and+dendritic+polymer+adhesives+for+corneal+laceration+repair+in+an+in+vivo+chicken+model.">Comparison of sutures and dendritic polymer adhesives for corneal laceration repair in an in vivo chicken model.</a> Archives of Ophthalmology. 127 (4), 442-447 (2009).</li><li>Fowler, W. C., Chang, D. H., Roberts, B. C., Zarovnaya, E. L., Proia, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+paradigm+for+corneal+wound+healing+research:+the+white+leghorn+chicken+(Gallus+gallus+domesticus).">A new paradigm for corneal wound healing research: the white leghorn chicken (Gallus gallus domesticus).</a> Current Eye Research. 28 (4), 241-250 (2004).</li><li>Huh, M. I., Kim, Y. E., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+TGF-&#946;+isoforms+and+signaling+intermediates+in+corneal+fibrotic+wound+repair.">The distribution of TGF-&#946; isoforms and signaling intermediates in corneal fibrotic wound repair.</a> Journal of Cellular Biochemistry. 108 (2), 476-488 (2009).</li><li>Spurlin, J. W., Lwigale, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounded+embryonic+corneas+exhibit+nonfibrotic+regeneration+and+complete+innervation.">Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation.</a> Investigative Ophthalmology &amp; Visual Science. 54 (9), 6334-6344 (2013).</li><li>Koudouna, E., Spurlin, J., Babushkina, A., Quantock, A. J., Jester, J. V., Lwigale, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulation+of+normal+collagen+architecture+in+embryonic+wounded+corneas.">Recapitulation of normal collagen architecture in embryonic wounded corneas.</a> Scientific Reports. 10 (1), 13815 (2020).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Developmental Dynamics. 233 (4), 1470-1477 (2005).</li><li>Spurlin, J., Lwigale, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations.">A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.</a> Developmental Dynamics. 242 (2), 148-154 (2013).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Neath, P., Roche, S. M., Bee, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+pressure+dependent+and+independent+growth+phases+of+the+embryonic+chick+eye+and+cornea.">Intraocular pressure dependent and independent growth phases of the embryonic chick eye and cornea.</a> Investigative Ophthalmology &amp; Visual Science. 32 (9), 2483-2491 (1991).</li><li>Matsuda, A., Yoshiki, A., Tagawa, Y., Matsuda, H., Kusakabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+wound+healing+in+tenascin+knockout+mouse.">Corneal wound healing in tenascin knockout mouse.</a> Investigative Ophthalmology &amp; Visual Science. 40 (6), 1071-1080 (1990).</li><li>Nishida, T., Nakagawa, S., Nishibayashi, C., Tanaka, H., Manabe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+enhancement+of+corneal+epithelial+wound+healing+of+rabbits+in+vivo.">Fibronectin enhancement of corneal epithelial wound healing of rabbits in vivo.</a> Archives of Ophthalmology. 102 (3), 455-456 (1984).</li><li>Sumioka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+cornea+wound+healing+in+a+tenascin+C-deficient+mouse+model.">Impaired cornea wound healing in a tenascin C-deficient mouse model.</a> Lab Investigation. 93 (2), 207-217 (2013).</li><li>Tervo, K., van Setten, G. B., Beuerman, R. W., Virtanen, I., Tarkkanen, A., Tervo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+tenascin+and+cellular+fibronectin+in+the+rabbit+cornea+after+anterior+keratectomy.+Immunohistochemical+study+of+wound+healing+dynamics.">Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics.</a> Investigative Ophthalmology &amp; Visual Science. 32 (11), 2912-2918 (1991).</li><li>Lwigale, P. Y., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens-derived+Semaphorin3A+regulates+sensory+innervation+of+the+cornea.">Lens-derived Semaphorin3A regulates sensory innervation of the cornea.</a> Developmental Biology. 306 (2), 750-759 (2007).</li><li>Kubilus, J. K., Linsenmayer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+corneal+innervation:+interactions+between+nerves+and+specialized+apical+corneal+epithelial+cells.">Developmental corneal innervation: interactions between nerves and specialized apical corneal epithelial cells.</a> Investigative Ophthalmology &amp; Visual Science. 51 (2), 782-789 (2010).</li><li>Schwend, T., Deaton, R. J., Zhang, Y., Caterson, B., Conrad, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+sulfated+glycosaminoglycans+and+their+effects+on+trigeminal+nerve+growth+cone+behavior+in+vitro:+roles+for+ECM+in+cornea+innervation.">Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation.</a> Investigative Ophthalmology &amp; Visual Science. 53 (13), 8118-8137 (2012).</li><li>Lee, M. K., Tuttle, J. B., Rebhun, L. I., Cleveland, D. W., Frankfurter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+and+posttranslational+modification+of+a+neuron-specific+beta-tubulin+isotype+during+chick+embryogenesis.">The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis.</a> Cell Motility and the Cytoskeleton. 17 (2), 118-132 (1990).</li><li>Chen, X., Nadiarynkh, O., Plotnikov, S., Campagnola, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+generation+microscopy+for+quantitative+analysis+of+collagen+fibrillar+structure.">Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure.</a> Nature Protocols. 7, 654-669 (2012).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+high-resolution+second-harmonic+generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.</a> Biophysical Journal. 82 (1), 493-508 (2002).</li><li>Cloney, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+ex-ovo+culturing+of+chick+embryos+to+advanced+stages+of+development.">Optimized ex-ovo culturing of chick embryos to advanced stages of development.</a> Journal of Visualized Experiments. (95), e52129 (2015).</li><li>Waldvogel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bird's+eye+view.">The bird's eye view.</a> American Scientist. 78, 342-353 (1990).</li><li>Martin, P., Parkhurst, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallels+between+tissue+repair+and+embryo+morphogenesis.">Parallels between tissue repair and embryo morphogenesis.</a> Development. 131 (13), 3021-3034 (2004).</li><li>Wilson, S. E., Mohan, R. R., Mohan, R. 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The authors have no competing financial interests concerning the information presented in this manuscript.

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&#160;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...

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 models1,2,5,6,7. 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 patterns8,9.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G hypodermic needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>30 degree angled microdissecting knife</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor labelled secondary antibodies</td>
			<td>Molecular Probes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2-H20)</td>
			<td>Sigma</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken egg trays</td>
			<td>GQF</td>
			<td>O246</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip, Serrated</td>
			<td>VWR</td>
			<td>82027-408</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors, sharp tip</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris 1 x 2 Teeth Tissue Forceps, Full Curved</td>
			<td>VWR</td>
			<td>100494-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Sigma</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting Scissors</td>
			<td>VWR</td>
			<td>470315-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-fibronectin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>B3/D6</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-laminin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3H11</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse antineuron-specific &#946;-tubulin (Tuj1, IgG2a)</td>
			<td>Biolegend</td>
			<td>801213</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-tenascin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>M1-B4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Sportsman 1502 egg incubator</td>
			<td>GQF</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tear by hand packaging (1.88 inch width)</td>
			<td>Scotch</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating scarless tissue regeneration in embryonic wounded chick corneas

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.

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...

Watch this Scientific Journal Video about Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas at JoVE.com

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

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.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

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.

investigating-scarless-tissue-regeneration-embryonic-wounded-chick

Research

JoVE Journal

Developmental Biology

1.8K visualizações.  Illinois Wesleyan University. Danos no estroma córnea adulto levam a uma resposta rápida de cicatrização de feridas que compromete a visão devido à formação opaca de tecido cicatricial. Este protocolo gera feridas em córneas de filhotes embrionários que, ao contrário dos adultos, podem curar sem cicatrizes. Dada a capacidade intrínseca da córnea embrionária ferida para se submeter a uma recapitulação completa da estrutura corneal nativa sem cicatrizes detectáveis, o filhote embrionário serve um important...