Name: zebrafish corneal wound healing: from abrasion to wound closure imaging analysis
Uploaded: 2022-03-01T15:00:00
Description: Watch this Scientific Journal Video about Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis at JoVE.com

The authors thank Pertti Panula for the access to the Zebrafish unit and Henri Koivula for the guidance and help with the zebrafish experiments. This research was supported by the Academy of Finland, the Jane and Aatos Erkko Foundation, the Finnish Cultural Foundation, and the ATIP-Avenir Program. Imaging was performed at the Electron Microscopy unit and the Light Microscopy Unit, Institute of Biotechnology, supported by HiLIFE and Biocenter Finland.

All experiments were approved by the national animal experiment board.
1. Preparations
<ol>
	<li>Prepare the tricaine stock solution used for anesthesia26 in advance (0.4% stock solution used in this protocol). Use gloves and keep the materials in a fume hood whenever possible.
	<ol>
		<li>For 50 mL of a 0.4% solution, weigh 200 mg of tricaine powder into a 50 mL tube. Dissolve the powder in approximately 45 mL of double-distilled water.</li>
		<li>Ad...

<ol>
	<li>Baden, T., Euler, T., Berens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+retinal+basis+of+vision+across+species.">Understanding the retinal basis of vision across species.</a> Nature Reviews.Neuroscience. 21 (1), 5-20 (2020).</li><li>Nishida, T., Saika, S., Morishige, N., Manis, M. J., Holland, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cornea+and+sclera:+Anatomy+and+physiology.">Cornea and sclera: Anatomy and physiology.</a> Cornea: Fundamentals, diagnosis and management, 4th ed. , 1-22 (2017).</li><li>Wilson, S. E., Liu, J. J., Mohan, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+interactions+in+the+cornea.">Stromal-epithelial interactions in the cornea.</a> Progress in Retinal and Eye Research. 18 (3), 293-309 (1999).</li><li>Wilson, S. E., 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=Epithelial+injury+induces+keratocyte+apoptosis:+hypothesized+role+for+the+interleukin-1+system+in+the+modulation+of+corneal+tissue+organization+and+wound+healing.">Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing.</a> Experimental Eye Research. 62 (4), 325-327 (1996).</li><li>Zieske, J. D., Guimaraes, S. R., Hutcheon, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+keratocyte+proliferation+in+response+to+epithelial+debridement.">Kinetics of keratocyte proliferation in response to epithelial debridement.</a> Experimental Eye Research. 72 (1), 33-39 (2001).</li><li>West-Mays, J. A., Dwivedi, D. 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+keratocyte:+corneal+stromal+cell+with+variable+repair+phenotypes.">The keratocyte: corneal stromal cell with variable repair phenotypes.</a> The International Journal of Biochemistry &amp; Cell Biology. 38 (10), 1625-1631 (2006).</li><li>Ahmed, F., House, R. J., Feldman, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+abrasions+and+corneal+foreign+bodies.">Corneal abrasions and corneal foreign bodies.</a> Primary Care. 42 (3), 363-375 (2015).</li><li>Ben-Eli, H., Erdinest, N., Solomon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+complications+of+chronic+eye+rubbing+in+ocular+allergy.">Pathogenesis and complications of chronic eye rubbing in ocular allergy.</a> Current Opinion in Allergy and Clinical Immunology. 19 (5), 526-534 (2019).</li><li>Wilson, S. A., Last, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+corneal+abrasions.">Management of corneal abrasions.</a> American Family Physician. 70 (1), 123-128 (2004).</li><li>Nagata, M., 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=JBP485+promotes+corneal+epithelial+wound+healing.">JBP485 promotes corneal epithelial wound healing.</a> Scientific Reports. 5, 14776 (2015).</li><li>Wang, X., 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=MANF+promotes+diabetic+corneal+epithelial+wound+healing+and+nerve+regeneration+by+attenuating+hyperglycemia-induced+endoplasmic+reticulum+stress.">MANF promotes diabetic corneal epithelial wound healing and nerve regeneration by attenuating hyperglycemia-induced endoplasmic reticulum stress.</a> Diabetes. 69 (6), 1264-1278 (2020).</li><li>Li, F. J., 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=Evaluation+of+the+AlgerBrush+II+rotating+burr+as+a+tool+for+inducing+ocular+surface+failure+in+the+New+Zealand+White+rabbit.">Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.</a> Experimental Eye Research. 147, 1-11 (2016).</li><li>Kalha, S., Kuony, A., Michon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+abrasion+with+ocular+burr+as+a+model+for+cornea+wound+healing.">Corneal epithelial abrasion with ocular burr as a model for cornea wound healing.</a> Journal of Visualized Experiments:JoVE. (137), e58071 (2018).</li><li>Kalha, S., 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=Bmi1++progenitor+cell+dynamics+in+murine+cornea+during+homeostasis+and+wound+healing.">Bmi1+ progenitor cell dynamics in murine cornea during homeostasis and wound healing.</a> Stem Cells. 36 (4), 562-573 (2018).</li><li>Park, M., 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=Visualizing+the+contribution+of+keratin-14(+)+limbal+epithelial+precursors+in+corneal+wound+healing.">Visualizing the contribution of keratin-14(+) limbal epithelial precursors in corneal wound healing.</a> Stem Cell Reports. 12 (1), 14-28 (2019).</li><li>Kuony, A., 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=Ectodysplasin-A+signaling+is+a+key+integrator+in+the+lacrimal+gland-cornea+feedback+loop.">Ectodysplasin-A signaling is a key integrator in the lacrimal gland-cornea feedback loop.</a> Development. 146 (14), (2019).</li><li>Farrelly, O., 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=Two-photon+live+imaging+of+single+corneal+stem+cells+reveals+compartmentalized+organization+of+the+limbal+niche.">Two-photon live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche.</a> Cell Stem Cell. 28 (7), 1233-1247 (2021).</li><li>Ikkala, K., Michon, F., Stratoulias, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+Zebrafish+corneal+injury+induces+bilateral+cell+plasticity+supporting+wound+closure.">Unilateral Zebrafish corneal injury induces bilateral cell plasticity supporting wound closure.</a> Scientific Reports. , (2021).</li><li>Ormerod, L. D., Abelson, M. B., Kenyon, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+models+of+corneal+injury+using+alkali-immersed+filter+discs.">Standard models of corneal injury using alkali-immersed filter discs.</a> Investigative Ophthalmology &amp; Visual Science. 30 (10), 2148-2153 (1989).</li><li>Anderson, C., Zhou, Q., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alkali-burn+injury+model+of+corneal+neovascularization+in+the+mouse.">An alkali-burn injury model of corneal neovascularization in the mouse.</a> Journal of visualized experiments: JoVE. (86), e51159 (2014).</li><li>Choi, H., 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=Comprehensive+modeling+of+corneal+alkali+injury+in+the+rat+eye.">Comprehensive modeling of corneal alkali injury in the rat eye.</a> Current Eye Research. 42 (10), 1348-1357 (2017).</li><li>Singh, P., Tyagi, M., Kumar, Y., Gupta, K. K., Sharma, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+chemical+injuries+and+their+management.">Ocular chemical injuries and their management.</a> Oman Journal of Ophthalmology. 6 (2), 83-86 (2013).</li><li>Pal-Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BALB/c+and+C57BL6+mouse+strains+vary+in+their+ability+to+heal+corneal+epithelial+debridement+wounds.">BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds.</a> Experimental Eye Research. 87 (5), 478-486 (2008).</li><li>Chen, J. J., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+corneal+epithelial+wound+healing+in+partial-thickness+removal+of+limbal+epithelium.">Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium.</a> Investigative Ophthalmology &amp; Visual Science. 32 (8), 2219-2233 (1991).</li><li>Xeroudaki, M., Peebo, B., Germundsson, J., Fagerholm, P., Lagali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGTA+in+corneal+wound+healing+after+transepithelial+laser+ablation+in+a+rabbit+model:+a+randomized,+blinded,+placebo-controlled+study.">RGTA in corneal wound healing after transepithelial laser ablation in a rabbit model: a randomized, blinded, placebo-controlled study.</a> Acta Ophthalmologica. 94 (7), 685-691 (2016).</li><li>. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio Available from: <a target="_blank" href="https://zfinorg/zf_info/zfbook/zfbk.html">https://zfinorg/zf_info/zfbook/zfbk.html</a> (2000)</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Xu, C., Volkery, S., Siekmann, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intubation-based+anesthesia+for+long-term+time-lapse+imaging+of+adult+zebrafish.">Intubation-based anesthesia for long-term time-lapse imaging of adult zebrafish.</a> Nature Protocols. 10 (12), 2064-2073 (2015).</li><li>Crosson, C. E., Klyce, S. D., Beuerman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+wound+closure+in+the+rabbit+cornea.+A+biphasic+process.">Epithelial wound closure in the rabbit cornea. A biphasic process.</a> Investigative Ophthalmology &amp; Visual Science. 27 (4), 464-473 (1986).</li><li>Parlanti, P., 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=Axonal+debris+accumulates+in+corneal+epithelial+cells+after+intraepithelial+corneal+nerves+are+damaged:+A+focused+Ion+Beam+Scanning+Electron+Microscopy+(FIB-SEM)+study.">Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study.</a> Experimental Eye Research. 194, 107998 (2020).</li><li>Zhao, X. C., 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=The+zebrafish+cornea:+structure+and+development.">The zebrafish cornea: structure and development.</a> Investigative Ophthalmology &amp; Visual Science. 47 (10), 4341-4348 (2006).</li><li>Richardson, R., 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=Re-epithelialization+of+cutaneous+wounds+in+adult+zebrafish+combines+mechanisms+of+wound+closure+in+embryonic+and+adult+mammals.">Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.</a> Development. 143 (12), 2077-2088 (2016).</li><li>van Loon, A. P., Erofeev, I. S., Maryshev, I. V., Goryachev, A. B., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+contraction+drives+the+3D+patterning+of+epithelial+cell+surfaces.">Cortical contraction drives the 3D patterning of epithelial cell surfaces.</a> The Journal of Cell Biology. 219 (3), (2020).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> Journal of Neurobiology. 44 (3), 289-307 (2000).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li><li>Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regrowth+after+spinal+cord+transection+in+adult+zebrafish.">Axonal regrowth after spinal cord transection in adult zebrafish.</a> The Journal of Comparative Neurology. 377 (4), 577-595 (1997).</li><li>Hu, X., 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=Sirt6+deficiency+impairs+corneal+epithelial+wound+healing.">Sirt6 deficiency impairs corneal epithelial wound healing.</a> Aging. 10 (8), 1932-1946 (2018).</li><li>Ksander, B. R., 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=ABCB5+is+a+limbal+stem+cell+gene+required+for+corneal+development+and+repair.">ABCB5 is a limbal stem cell gene required for corneal development and repair.</a> Nature. 511 (7509), 353-357 (2014).</li><li>Pan, Y. A., 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=Zebrabow:+multispectral+cell+labeling+for+cell+tracing+and+lineage+analysis+in+zebrafish.">Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish.</a> Development. 140 (13), 2835-2846 (2013).</li></ol>

Corneal physical injuries are the most common cause of ophthalmology patient visits to the hospital. Therefore, it is important to establish relevant models for the study of different aspects of corneal pathophysiology. So far, the mouse is the most commonly used model for the study of corneal wound healing. However, adding eyedrops on murine wounded eyes to validate the impact of specific drugs on corneal wound healing can be difficult. In this respect, the zebrafish model is particularly useful for the pharmacological ...

As most of the epithelia are in contact with the external environment, they are prone to physical injury, making them well suited for the study of wound healing processes. Among the well-studied tissues, the cornea is an extremely useful model in the investigation of the cellular and molecular aspects of wound healing. As a transparent external surface, it provides physical protection to the eye and is the first element to focus the light onto the retina. While the structure and cell composition of the retina differ between species1, these elements of the cornea are generally similar in all camera-type eyes, regardless of species.
The cornea is composed of three main layers2. The first and outermost layer is the epithelium, which is constantly renewed to ensure its transparency. The second layer is the stroma, which contains scattered cells, called keratocytes, within a thick layer of strictly organized collagen fibers. The third and innermost layer is the endothelium, which allows nutrient and liquid diffusion from the anterior chamber to the outer layers. The epithelial and stromal cells interact via growth factors and cytokines3. This interaction is highlighted by the rapid apoptosis and subsequent proliferation of keratocytes after epithelial injury4,5. In case of a deeper wound, such as a puncture, keratocytes take an active part in the healing process6.
Being in contact with the external environment, corneal physical injuries are common. Many of them are caused by small foreign objects7, such as sand or dust. The reflex of eye rubbing can lead to extensive epithelial abrasions and corneal remodelling8. According to wound size and depth, these physical injuries are painful and take several days to heal9. The optimal wound healing characteristics of a model facilitate the understanding of the cellular and molecular aspects of wound closure. Furthermore, such models have also proved useful for testing new molecules with the potential to accelerate corneal healing, as previously demonstrated10,11.
The protocol described here aims to use zebrafish as a relevant model to study corneal physical injury. This model is highly convenient for pharmacological screening studies as it allows molecules to be added directly to the tank water and, therefore, to come into contact with a healing cornea. The details provided here will help scientists perform their studies on the zebrafish model. The in vivo injury is performed with a dulled ocular burr. The impact on epithelial cells adjoining or at a distance from it can be analyzed by specifically removing the central corneal epithelium. In recent years, numerous reports focused on such a method on rodent cornea12,13,14,15,16,17; however, to date, only a single report has applied this method to zebrafish18.
Because of its simplicity, the physical wound is useful in delineating the role of epithelial cells in wound closure. Another well-established model of corneal injury is the chemical burn, especially the alkali burn19,20,21. However, such an approach indirectly damages the entire eye surface, including the peripheral cornea and corneal stroma19. Indeed, alkali burns potentially induce corneal ulcers, perforations, epithelial opacification, and swift neovascularization22, and the uncontrollable outcome of alkali burns disqualifies that approach for general wound healing studies. Numerous other methods are also used to investigate corneal wound healing according to the particular focus of the study in question (e.g., complete epithelial debridement23, the combination of chemical and mechanical injury for partial-thickness wound24, excimer laser ablation for wounds extending to the stroma25). The use of an ocular burr restricts the focal point to the epithelial response to the wound and provides a highly reproducible wound.
As with each method of wound infliction, the use of an ocular burr has advantages and disadvantages. The main disadvantage is that the response being mostly epithelial, it does not perfectly reflect the abrasions seen in the clinical setting. However, this method has numerous advantages, including the ease with which it can be set up and performed, its precision, its reproducibility, and the fact that it is noninvasive, making it a method well tolerated by animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.1M Na-PO4 (sodium phosphate buffer), pH 7.4</td>
			<td >in-house</td>
			<td ></td>
			<td >Solution is prepared from 1M sodium phosphate buffer (1M Na2HPO4 adjusted to pH 7.4 with 1M NaH2PO4).</td>
		</tr>
		<tr >
			<td >0.2M Na-PO4 (sodium phosphate buffer), pH 7.4</td>
			<td >in-house</td>
			<td ></td>
			<td >Solution is prepared from 1M sodium phosphate buffer (1M Na2HPO4 adjusted to pH 7.4 with 1M NaH2PO4).</td>
		</tr>
		<tr >
			<td >0.5mm burr tips</td>
			<td >Alger Equipment Company</td>
			<td >BU-5S</td>
			<td></td>
		</tr>
		<tr >
			<td >1M Tris, pH 8.8</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >adhesive tabs</td>
			<td >Agar Scientific</td>
			<td >G3347N</td>
			<td></td>
		</tr>
		<tr >
			<td >Algerbrush burr, Complete instrument</td>
			<td >Alger Equipment Company</td>
			<td >BR2-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Cotton swaps</td>
			<td >Heinz Herenz Hamburg</td>
			<td >1030128</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting plate</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting tools</td>
			<td >Fine Science Tools</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >double-distilled water</td>
			<td >in-house</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Eppedorf tubes, 2ml</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td >Sigma</td>
			<td >A5040</td>
			<td>Caution: causes irritation.</td>
		</tr>
		<tr >
			<td >Glutaraldehyde, 50% aqueous solution, grade I</td>
			<td >Sigma</td>
			<td >G7651</td>
			<td>Caution: toxic.</td>
		</tr>
		<tr >
			<td >Lidocaine hydrochloride</td>
			<td >Sigma</td>
			<td >L5647</td>
			<td>Caution: toxic.</td>
		</tr>
		<tr >
			<td >mounts</td>
			<td >Agar Scientific</td>
			<td >G301P</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td >Thermo Scientific</td>
			<td >101VR20</td>
			<td></td>
		</tr>
		<tr >
			<td >pH indicator strips</td>
			<td >Macherey-Nagel</td>
			<td >92110</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic spoons</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic tubes, 15 ml</td>
			<td >Greiner Bio-One</td>
			<td >188271</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic tubes, 50 ml</td>
			<td >Greiner Bio-One</td>
			<td >227261</td>
			<td></td>
		</tr>
		<tr >
			<td >Scanning electron microscope</td>
			<td >FEI</td>
			<td >Quanta 250 FEG</td>
			<td></td>
		</tr>
		<tr >
			<td >Soft sponge</td>
			<td >any provider</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sputter coater</td>
			<td >Quorum Technologies</td>
			<td >GQ150TS</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >Leica</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish corneal wound healing: from abrasion to wound closure imaging analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This study describes a method using an ophthalmic burr in zebrafish corneal wound healing experiments. The method is modified from previous studies on mice, where the burr was shown to remove the epithelial cell layers efficiently13. The challenges in zebrafish corneal wounding include the relatively small size of the eye, and in the case of time-consuming experiments, the need to maintain a constant water flow through the gills (as described by Xu and colleagues28). The ma...

Watch this Scientific Journal Video about Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis at JoVE.com

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Research

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Biology

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