Name: corneal epithelial abrasion with ocular burr as a model for cornea wound healing
Uploaded: 2018-07-10T14:30:00
Description: Watch this Scientific Journal Video about Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing at JoVE.com

We would like to thank Kaisa Ikkala for her invaluable technical assistance and insightful help when actualizing this method as well as later on during implementation to our central research questions. We would also like to thank the Laboratory Animal Center and Anna Meller for her help with planning the guidelines of the veterinary work.

All experiments are approved by the national animal experiment board.
1. Preparations
<ol>
	<li>Prepare all solutions and keep in room temperature, unless otherwise indicated. Follow waste disposal regulations to dispose used materials and solutions.</li>
	<li>Use NMRI and ICR outbred stocks, between ages 4-12 weeks and either gender. If using the C57BL/6 strain, follow the ketamine-medetomidine preparation method in step 1.3.2. For other steps, follow the instructions described in 1.3.3.-1....

<ol>
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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=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Experimental Eye Research. 121, 178-193 (2014).</li><li>Kalha, S., Shrestha, B., Sanz Navarro, M., Jones, K. B., Klein, O. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Thrombospondin-1+in+Repair+of+Penetrating+Corneal+Wounds.">Role of Thrombospondin-1 in Repair of Penetrating Corneal Wounds.</a> Investigative Ophthalmology &amp; Visual Science. 54 (9), 6262 (2013).</li><li>Gipson, I. K., Kiorpes, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+sheet+movement:+Protein+and+glycoprotein+synthesis.">Epithelial sheet movement: Protein and glycoprotein synthesis.</a> Developmental Biology. 92 (1), 259-262 (1982).</li><li>Danjo, Y., Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+&amp;#34;purse+string&amp;#34;+filaments+are+anchored+by+E-cadherin-mediated+adherens+junctions+at+the+leading+edge+of+the+epithelial+wound,+providing+coordinated+cell+movement.">Actin &amp;#34;purse string&amp;#34; filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement.</a> Journal of Cell Science. 111, 3323-3332 (1998).</li><li>Lyu, J., Joo, C. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt-7a+up-regulates+matrix+metalloproteinase-12+expression+and+promotes+cell+proliferation+in+corneal+epithelial+cells+during+wound+healing.">Wnt-7a up-regulates matrix metalloproteinase-12 expression and promotes cell proliferation in corneal epithelial cells during wound healing.</a> Journal of Biological Chemistry. 280 (22), 21653-21660 (2005).</li><li>Nagata, M., Nakamura, T., Hata, Y., Yamaguchi, S., Kaku, T., Kinoshita, S. <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> Science Reports. 5, 14776 (2015).</li><li>Stepp, M. A., Zhu, L., Cranfill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+beta+4+integrin+expression+and+localization+in+vivo+in+response+to+corneal+epithelial+injury.">Changes in beta 4 integrin expression and localization in vivo in response to corneal epithelial injury.</a> Investigative Ophthalmology &amp; Visual Science. 37 (8), 1593-1601 (1996).</li><li>Stepp, M. A., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+alpha+9+integrin+and+tenascin+during+epithelial+regeneration+after+debridement+in+the+cornea.">Upregulation of alpha 9 integrin and tenascin during epithelial regeneration after debridement in the cornea.</a> Journal of Histochemistry &amp; Cytochemistry. 45 (2), 189-201 (1997).</li><li>Pal-Ghosh, S., Tadvalkar, G., Jurjus, R. A., Zieske, J. D., Stepp, M. A. <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>. Lab Animal Research. Rodent Handling and Restraint Techniques Available from: <a target="_blank" href="https://www.jove.com/science-education/10221/rodent-handling-and-restraint-techniques">https://www.jove.com/science-education/10221/rodent-handling-and-restraint-techniques</a> (2018)</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+basement+membrane+enhances+corneal+wound+healing.">Removal of the basement membrane enhances corneal wound healing.</a> Experimental Eye Research. 93 (6), 927-936 (2011).</li><li>Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+and+cell-cell+interactions+during+corneal+epithelial+wound+healing.">Cell-matrix and cell-cell interactions during corneal epithelial wound healing.</a> Progress in Retinal and Eye Research. 22 (2), 113-133 (2003).</li><li>Sato, Y., Seo, N., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+differences+between+FVB+and+C57BL/6+mice+affect+hypnotic+susceptibility+to+pentobarbital,+ketamine+and+nitrous+oxide,+but+not+isoflurane.">Genetic background differences between FVB and C57BL/6 mice affect hypnotic susceptibility to pentobarbital, ketamine and nitrous oxide, but not isoflurane.</a> Acta Anaesthesiologica Scandinavica. 50 (5), 553-556 (2006).</li><li>Pajoohesh-Ganji, A., Pal-Ghosh, S., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K14+++Compound+niches+are+present+on+the+mouse+cornea+early+after+birth+and+expand+after+debridement+wounds.">K14 + Compound niches are present on the mouse cornea early after birth and expand after debridement wounds.</a> Developmental Dynamics. 245 (2), 132-143 (2016).</li><li>Boote, 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=Quantitative+Assessment+of+Ultrastructure+and+Light+Scatter+in+Mouse+Corneal+Debridement+Wounds.">Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds.</a> Investigative Ophthalmology &amp; Visual Science. 53 (6), 2786 (2012).</li><li>Pal-Ghosh, 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=MMP9+cleavage+of+the+&#946;4+integrin+ectodomain+leads+to+recurrent+epithelial+erosions+in+mice.">MMP9 cleavage of the &#946;4 integrin ectodomain leads to recurrent epithelial erosions in mice.</a> Journal of Cell Science. 124 (Pt 15), 2666-2675 (2011).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M., Stepp, M. 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Wounding methods are popular tools to study different aspects of corneal homeostasis and pathologies. The abrasion model offers a well-controlled method to address relevant problems in ophthalmology. However, certain critical points in the protocol are worth emphasizing. Notably, the details outlined regarding the veterinary medicine, wound healing timeline, and outcome are optimized for use with outbred NMRI and ICR stocks, but may vary among strains of mice26. With this protocol, the experimenta...

Epithelial layers of numerous organs are exposed to injuries. However, they also contain the ability to compensate for tissue loss through wound healing. The cornea offers an excellent model to study wound healing. It forms the external surface of the eye and provides a protective layer for the sensitive ocular machinery. Thus, cornea functions as a physical barrier to pathogens and water loss. It is composed of three layers; epithelium, stroma and endothelium. The epithelium of the cornea makes up the outermost layer of the cornea. Epithelial cells maintain the barrier function of the cornea by adhering strictly to each other through tight junctions1,2,3. An acellular corneal basement membrane, the Bowman&#39;s membrane, separates the epithelium from the extensive stroma, which contains refractory keratocytes. Under the stroma, endothelial cells channel nutrients, water, and oxygen to the upper layer.
Corneal abrasions are very common in the clinic4. Injuries to the cornea are diverse, but are largely caused by small particles such as dust or sand, scratches, or other foreign objects. The protocol described here aims at reproducing a clinically relevant type of corneal epithelial abrasion. In doing so, this protocol provides a controllable and seminal method for clinicians and corneal scientists to implement in their own studies. We have performed an in vivo injury repair assay on the murine cornea by abrading the tissue with a dulled ocular burr, the Algerbrush II. Here, we target the abrasion only to the central corneal epithelium and leave the other parts of the organ without damage. Thus, the protocol is ideal to study corneal epithelial cell dynamics or the basement membrane during re-epithelialization, cell migration, proliferation and differentiation in vivo5. Recently, this model was used to analyze progenitor cell dynamics in the murine cornea as well as to unveil the capacity of the differentiated corneal epithelial cells in re-establishing the corneal stem cell niche after injury6,7. Following abrasion, the cornea returns to its normal transparency and tensile strength. Interestingly, an in vitro study indicated that re-epithelialization occurs without increased cell proliferation8. This protocol describes the timeline of uninterrupted healing in the murine cornea. The method is thus applicable to test the effect of drugs on healing patterns and speed.
The cornea has been extensively used for wound healing studies. However, many studies have relied on other models of injury. A well-established model of corneal injury is the alkaline burn that is performed by applying sodium hydroxide (NaOH) with or without filter paper on the corneal surface9. Alkaline exposure results in a large and diffuse injury that affects not only the corneal epithelium, but also the conjunctiva and stroma9,10. Strong alkaline solutions have been shown to induce corneal ulcers, opacification, and neovascularization9. Inflammatory cells invade the stroma typically within 6 h and remain there until 24 h11. Thus, alkaline injury is an advisable method in studies related to stromal activation. Another type of chemical injury can be inflicted by applying dimethyl sulfoxide (DMSO) on the cornea9,10. Other commonly used injury models include incisional wounds that penetrate through the stroma and keratectomy wounds, which are limited to the upper portion of the stroma14,15. These methods are also useful to answer questions regarding stromal wound healing. Different injury models have their own advantages and disadvantages. Abrasion, or debridement, of the corneal epithelium was first developed using dulled scalpels or blades on ex vivo corneas16. This method has later been used in vivo on mouse, rat, and rabbit17,18,19,20,21,22. Using the ocular burr (Figure 1), we remove only a selected region of the epithelium, leaving the rest of the epithelium unaffected. This way, it is possible to precisely target the epithelial removal to different parts of the cornea. In addition, the abrasion size can be assessed with fluorescein staining. Furthermore, here we follow abrasion closure during the healing period.
This method poses several advantages, i) including precise location of abrasion site, which is not possible with chemical injury, ii) the abrasion is quick to perform, and iii) it is non-invasive. Herein, we describe the method using the outbred NMRI mouse as a model, however this could be applied to the vast array of mouse genetic models, as well as to the rat and rabbit, which are common models used to study human corneal disruption.

<table>
	<tbody>
		<tr>
			<td>NMRI mouse</td>
			<td>Envigo</td>
			<td>275</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% NaCl</td>
			<td></td>
			<td></td>
			<td>use sterile</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Vetmedic</td>
			<td>Vnr087896</td>
			<td>Market name: Cepetor Vet</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td>Vnr511485</td>
			<td>Market name: Ketaminol Vet</td>
		</tr>
		<tr>
			<td>Buprenorfin</td>
			<td>Invidior</td>
			<td>3015248</td>
			<td>Market name: Temgesic</td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Orion Pharma</td>
			<td>Vnr471953</td>
			<td>Market name: Antisedan Vet</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vnr027579</td>
			<td>Market name: Norocarp Vet</td>
		</tr>
		<tr>
			<td>1% fucidin acid eye ointment</td>
			<td>Dechra</td>
			<td>Vnr080899</td>
			<td>Market name: Isathal</td>
		</tr>
		<tr>
			<td>Fluorescein salt</td>
			<td>Sigma-Aldrich</td>
			<td>F6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline solution</td>
			<td></td>
			<td></td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Algerbrush ii ocular burr (0.5 mm tip)</td>
			<td>Algerbrush</td>
			<td>6.39768E+11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt Blue pen light</td>
			<td>SP Services</td>
			<td>DE/003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Kunz Instruments</td>
			<td>2007-0217</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital SLR camera</td>
			<td>Nikon</td>
			<td>D80</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable camera arm and clamp</td>
			<td>Neewer</td>
			<td>10086132</td>
			<td>Height 28 cm</td>
		</tr>
		<tr>
			<td>Table lamp with a flexible arm and a clamp</td>
			<td>Prisma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soft wipe</td>
			<td>KimtechScience</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection toolset</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Beckton Dickinson</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles</td>
			<td>Beckton Dickinson</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Eppendorf tube</td>
			<td>Sarstedt</td>
			<td>689</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue casette</td>
			<td>Sakura Finetech</td>
			<td>4118F</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processing machine ASP200S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>UN1307</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Millipore</td>
			<td>K95523361</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding mold 32 x 25 x 6 mm</td>
			<td>Sakura Finetech</td>
			<td>4123</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM355</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for sectioning</td>
			<td>Orthex</td>
			<td>60591</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for sectioning</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Feather</td>
			<td>S35</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Th.Geyer GmbH &#38; Co.</td>
			<td>7,695,019</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Millipore</td>
			<td>MPGP04001</td>
			<td>MilliQ</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>PFA</td>
		</tr>
	</tbody>
</table>

corneal epithelial abrasion with ocular burr as a model for cornea wound healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a model to inflict an abrasion injury to the mouse cornea and suggests how to follow and visualize the healing process after abrasion. Recently, we employed this method to study the role of corneal epithelial progenitor cells during wound healing6. The use of established tools is the key to a successful abrasion experiment. We, and others, have used the Algerbrush II ocular burr (Figure 1) to perform the abrasio...

Watch this Scientific Journal Video about Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing at JoVE.com

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing (Video) | JoVE

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

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Medicine

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