We are grateful for Dr. Xinyu Li's help in preparing the manuscript. S.W. was supported by a Startup fund from Tulane University, President's Research Council New Investigator Award from UT Southwestern Medical Center, NIH Grant EY021862, a career development award from the Research to Prevent Blindness foundation, and a Bright Focus Award in Age Related Macular Degeneration Research.

Note: The following protocol and representative results use the HDAC inhibitor SAHA as an example compound. However, this protocol is by no means limited to the use of SAHA, and is recommended as a general method to test the effects of soluble compounds on corneal neovascularization. Minor modifications will need to be made for degree of dilution as well as frequency and duration of application. Additionally, compounds that are easily soluble in water will be able to be administered in the absence of DMSO.
<p class="...

<ol>
	<li>Pascolini, D., Mariotti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment.">Global estimates of visual impairment.</a> Br. J. Ophthalmol. 96 (5), 614-618 (2010).</li><li>Whitcher, J., Srinivasan, M., Upadhyay, 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+Blindness:+A+Global+Perspective.">Corneal Blindness: A Global Perspective.</a> Bull. World Health Org. 79 (3), 214-221 (2003).</li><li>Gupta, D., Illingworth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatments+for+corneal+neovascularization:+a+review.">Treatments for corneal neovascularization: a review.</a> Cornea. 30 (8), 927-938 (2011).</li><li>Cursiefen, 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=Consensus+statement+on+indications+for+anti-angiogenic+therapy+in+the+management+of+corneal+diseases+associated+with+neovascularisation:+outcome+of+an+expert+roundtable.">Consensus statement on indications for anti-angiogenic therapy in the management of corneal diseases associated with neovascularisation: outcome of an expert roundtable.</a> Br. J. Ophthalmol. 96 (1), 3-9 (2012).</li><li>Delmonte, D., 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=Anatomy+and+Physiology+of+the+Cornea.">Anatomy and Physiology of the Cornea.</a> J. Cataract Refract. Surg. 37 (3), 588-598 (2011).</li><li>Cursiefen, C., Seitz, B., Dana, M. R., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+lymphangiogenesis+in+the+cornea.+Pathogenesis,+clinical+implications+and+treatment+options.">Angiogenesis and lymphangiogenesis in the cornea. Pathogenesis, clinical implications and treatment options.</a> Der Ophthalmologe. 100 (4), 292-229 (2003).</li><li>Chang, J., Gabison, E., Kato, T., Azar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Neovascularization.">Corneal Neovascularization.</a> Curr. 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The protocol presented here results in reproducible levels of hemangiogenesis, lymphangiogenesis, and inflammation, making it an ideal system to study these three (interrelated) processes. While this method produces centralized corneal NV, several methods that have been developed to cause more directed NV, namely suturing of the cornea17 and implanted growth-factor expressing pellets18, might also be of interest. Our protocol is designed for use in the adult mouse, providing an&#160;easy to use anim...

Corneal blindness is the fourth most common cause of blindness, responsible for approximately 4% of all cases1. Corneal neovascularization (NV) plays a significant role in many of these pathologies, including herpetic keratitis (the leading infectious cause of blindness in the West) and trachoma (the leading cause of infectious blindness worldwide)2. Current therapies include steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-VEGF therapies, and cyclosporin A as well as conventional or laser surgical techniques3. However, the severely debilitating nature of corneal NV based pathologies, the paucity of surgical facilities capable of treating corneal NV, and the lack of a strongly performing pharmacological option led a recent expert roundtable to conclude that, despite the extant therapies, the fundamental medical need presented by these pathologies remains unmet4.
The human cornea consists of 5 layers, 3 cellular layers (epithelial, stromal and endothelium) and 2 interface (Bowman membrane and Descemet membrane). It functions as a mechanical barrier and refractive surface for the eye. Its transparent nature is the consequence of a delicate balance of its components and is integral to its proper function5. Normally avascular, the cornea receives blood from microvessels running along its outer edge which are fed from the ciliary and ophthalmic arteries. Corneal NV occurs when a stimulus promotes angiogenesis of these vessels allowing them to grow towards the center of the cornea and thus limit vision6. Corneal angiogenesis includes hemangiogenesis and lymphangiogenesis, which result in the ingrowth of blood vessels and lymphatic vessels from the limbal vascular arcade towards the center of the cornea. This leads to a breakdown of corneal &#34;angiogenic privilege&#34;, an increase in corneal opacity and fibrosis, disruption of the corneal layers, and edema7. The precise triggers of corneal NV are numerous, ranging from a response to infectious disease such as trachoma to a chemically induced state caused traditional medicines, industrial chemicals, or even chemical warfare agents.
The molecular mechanisms of this process are not, as yet, fully characterized; however, a few key players have been identified. Under normal conditions the cornea possesses a unique &#8216;angiogenic privilege&#39; maintained by a redundant array of anti-angiogenic factors (such as soluble VEGF-R1)8. However, in response to an external stimulus (such as an injury), there will be a local upregulation of pro-angiogenic factors (e.g. VEGF-A). This tips the balance of pro and anti- angiogenic factors that underlies the cornea&#39;s angiogenic privilege, and leads to hemangiogenesis, lymphangeogenesis, and inflammation, therefore causing corneal pathology and even blindness9.
Given the unmet medical need of this highly debilitating pathology, it is of interest to the field to have a reliable animal model of corneal NV. Here we present such a model: controlled alkali-burn injury. Various eye-injury models based on using filter paper rings have been used since 1970s10. In 1989, a group of Harvard Medical School ophthalmologists characterized a standard model of a central corneal alkali-burn injury in rabbit based on soaking a piece of circular filter paper with sodium hydroxide (NaOH) and applying it to the cornea at a specific range of concentrations11. Since then, this technique has been adapted for use in the mouse12-14. Recently, the Wang lab studied the therapeutic effects of the histone deacetylase (HDAC) inhibitor SAHA in the pathogenesis of corneal NV using a mouse corneal alkali-burn injury model15. The methodology of the mouse corneal alkali-burn injury model presented here was built mainly on the prior work of two other papers14,16.

<table border="0" cellpadding="0" cellspacing="0" width="598">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">309659</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">306575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25 Guage Needle</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">305916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-F4/80 (rat anti-mouse)</td>
			<td style="width:96px;">AbD Serotech</td>
			<td>MCA497RT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-LYVE-1 (rabbit anti-mouse)</td>
			<td style="width:96px;">Abcam</td>
			<td style="width:119px;">ab14917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-PECAM-1 (rat anti-mouse)</td>
			<td style="width:96px;">BD</td>
			<td align="right" style="width:119px;">553370</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa488 (goat anti-rat)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-IgG Alexa594 (goat anti-rabbit)</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">A11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:96px;">Tucsen</td>
			<td style="width:119px;">TCC 5.0 ICE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-548-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D4540-1L</td>
			<td>Caution: Mutagenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Blunt), Iris</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps (Sharp), Dumont #4</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">500340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td>RXKETAMINE</td>
			<td>Controlled substance, proper license required for use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Source for Microscope</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">LED-14M-YA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope (Stereo 7X-45X)</td>
			<td style="width:96px;">AmScope</td>
			<td style="width:119px;">SM-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mounting Medium, Vectashield</td>
			<td style="width:96px;">Vector</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S397-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">S318-1</td>
			<td>Caution: Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:96px;"></td>
			<td style="width:119px;">P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proparacaine Hydrochloride</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">P4554-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors (5mm blade), Vanas</td>
			<td style="width:96px;">WPI</td>
			<td align="right" style="width:119px;">14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat Serum</td>
			<td style="width:96px;">MPBio</td>
			<td align="right" style="width:119px;">92939249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope Slides</td>
			<td style="width:96px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">T8787-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whatman Grade 1 Filter Paper</td>
			<td style="width:96px;">Whatman</td>
			<td style="width:119px;">1001-6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Solution</td>
			<td style="width:96px;">MedVet</td>
			<td style="width:119px;">RXANASED-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

an alkali-burn injury model of corneal neovascularization in the mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

After alkali-burn injury, corneal NV occurs in a predictable, time-dependent fashion. Figure 1 demonstrates the stark difference both in neovascularization and corneal opacity between an untreated animal (Figure 1A) and an animal treated with the HDAC inhibitor SAHA (Figure 1B) at the 7 day time point.
Figures 2A and&#160;2B demonstrate a corneal flat mount of an untreated control eye with primary PECAM-1 and ...

Watch this Scientific Journal Video about An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse at JoVE.com

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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16.3K vues.  Tulane University.  Néovascularisation (NV) de la cornée peut compliquer multiples pathologies visuelles. Utilisant un modèle de lésion alcalin brûlage dirigé, un niveau quantifiable de la SA de la cornée peut être produite pour l'étude mécaniste de la SA de la cornée et l'évaluation des thérapies potentielles pour les troubles néovasculaire. 