This work was supported by the SRB Charitable Corporation, National Institutes of Health (NIH) P30EY002520, and an unrestricted institutional grant from Research to Prevent Blindness (RPB) to the Department of Ophthalmology, Baylor College of Medicine. W.L. is supported by The Knights Templar Eye Foundation Endowment in Ophthalmology.

All the animal work, including the experimental procedures and euthanasia, was approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine with Protocol Number AN-8790.
1. Preparation of 1 N NaOH
<ol>
	<li>Add 4 mL of sterile deionized water to a 15 mL centrifuge tube. Weigh out 400 mg of sodium hydroxide (NaOH), and add to the tube carefully.</li>
	<li>Dissolve the NaOH by slowly stirring the solution using a glass rod. Make up the volume to 10 mL by adding sterile deionized water to the tube, and mix again by gently inverting the tube up and down. Close the cap tightly, and store the solution at room temperature.</li>
	<li>Prepare the fresh solution every month because the concentration of the NaOH solution may be reduced by the solution absorbing carbon dioxide in the air.</li>
	<li>Always gently mix the NaOH solution before use. 
	&#8203;CAUTION: Prepare the solution inside a chemical hood, and wear appropriate personal protective equipment (PPE).</li>
</ol>
2. Preparation of the 4% paraformaldehyde (PFA) solution
<ol>
	<li>Add 30 mL of 1x phosphate-buffered saline (PBS) to a glass beaker. Weigh out 4 g of paraformaldehyde (PFA), and add it to the beaker.</li>
	<li>Keep the beaker on a hot plate at 60 &#176;C with stirring. Add the 1 N NaOH solution dropwise to raise the pH until the solution clears.</li>
	<li>Check and adjust the pH to 7.4 using 1 N hydrochloric acid (HCl). Adjust the final volume to 50 mL with 1x PBS.</li>
	<li>Cool and filter the solution. Store the solution at 4 &#176;C. 
	&#8203;CAUTION: Prepare the solution in a fume hood while wearing appropriate PPE.</li>
</ol>
3. Preparing the ketamine/xylazine cocktail
<ol>
	<li>Prepare the ketamine/xylazine cocktail by adding 0.8 mL of ketamine (stock concentration: 100 mg/mL) and 0.16 mL of xylazine (stock concentration: 100 mg/mL) to 9.4 mL of saline.</li>
	<li>Store the cocktail in sterile injection bottles at room temperature (RT).</li>
</ol>
4. Alkali burn on the mouse cornea
<ol>
	<li>Inject meloxicam (4-6 mg/kg of body weight) subcutaneously 30 min prior to the procedure for pain relief. Anesthetize the mice (C57BL/6J, 6-8 weeks of age, male) using an i.p. injection of the ketamine/xylazine cocktail (Ketamine 80 mg/kg and Xylazine 16 mg/kg of body weight).</li>
	<li>Check the reflex response (pedal withdrawal) by pinching the toes of the mouse, and confirm the absence of the reflex. Apply one drop of topical anesthetic, 0.5% proparacaine, on the corneal surface of one eye, and a drop of artificial tears on the other eye.</li>
	<li>Using a 2 mm biopsy punch, punch out Whatman filter paper discs.</li>
	<li>Add 2 &#181;L of 1N NaOH to a clean Petri dish. Put the 2 mm filter paper disc on the 1 N NaOH drop, and allow it to soak for 15 s.</li>
	<li>Pick up the filter paper with forceps, and apply the filter paper to the proparacaine-treated eye at the center of the cornea for 30 s. 
	NOTE: The filter paper must be touching only the center of the cornea, and care must be taken to avoid the movement of the filter paper once placed, as moving the filter paper can cause burns to the adjacent tissues.</li>
	<li>Wash the eye by flushing with 20 mL of sterile saline solution in a sterile syringe. 
	NOTE: Care must be taken to ensure that&#160;the cornea, along with the conjunctival sac, is washed thoroughly to ensure no further damage to the cornea or surrounding tissue. Washing the conjunctival sac will further prevent symblepharon.</li>
	<li>Wipe the excess saline gently from the eyes and the surrounding area using disposable soft wipes. Afterward, keep the mice in a recovery cage on a warm heating pad until ambulatory. 
	NOTE: Mice are monitored daily after the alkali burn for 3 days. If symptoms of pain or stress are observed, meloxicam (4-6 mg/kg of body weight) is administered subcutaneously.</li>
</ol>
5. Examination and assessment of neovascularization and opacity
<ol>
	<li>In anesthetized mice, examine the eyes under a dissection microscope on day 10 after the burn, and obtain images using a camera attached to the dissection scope to score the opacity and neovascularization. 
	NOTE: A regular dissection scope with a camera attached is sufficient.</li>
	<li>While observing the cornea through the dissection microscope, score the opacity after the burn based on the following scale12: 
	0&#160;= No opacity; clear cornea 
	1&#160;= Mild opacity; slight haziness in the iris and pupil areas; iris and pupil easily visible 
	2&#160;= Moderate opacity; iris and pupil barely visible 
	3&#160;= Severe opacity; iris or pupil not visible 
	4&#160;= Opaque cornea; iris and pupil not visible</li>
	<li>While observing the cornea through the dissection microscope, score the CoNV based on the following scale12: 
	0&#160;= No neovascularization; no new vessels from the limbus 
	1&#160;= Mild neovascularization; new vessels originating from the limbus 
	2&#160;= Moderate neovascularization; blood vessels originated from the limbus and growing toward the center of the cornea 
	3&#160;= Severe neovascularization; blood vessels originating from the limbus and reaching and/or crossing the center of the cornea</li>
	<li>Use a Student&#39;s t-test to statistically compare the opacity and neovascularization scores between the alkali burn and healthy eye groups.</li>
	<li>Euthanize the mice on day 10 by isoflurane exposure at 5% until 1 min after breathing stops, followed by cervical dislocation, and collect the corneas for flat-mount imaging.</li>
</ol>
6. Optical coherence tomography (OCT) imaging
<ol>
	<li>Take OCT images of the anterior segment of the eyes in anesthetized mice on day 10 after the burn. Perform OCT image acquisition as a volume scan using IR + OCT mode with a 30&#176; field of view and 100% IR intensity.</li>
	<li>Quantify the thickness of the corneas using ImageJ software.</li>
	<li>To measure the thickness, use the Line Selection tool in ImageJ software to create a straight line between the anterior and posterior surfaces at the central cornea.</li>
	<li>Click on Analyze &#62; Measure in the software tools to transfer the values to the data window.</li>
	<li>Copy the values to a spreadsheet file, and statistically compare the corneal thickness between the alkali burn and healthy eye groups using a Student&#39;s t-test. 
	&#8203;NOTE: The thickness of the cornea is the distance from a point on the anterior corneal surface to the closest point on the posterior corneal surface at the corneal center.</li>
</ol>
7. Immunostaining for CoNV on flat-mount corneas
<ol>
	<li>Euthanize the mice on day 10 post alkali burn, and enucleate the eyes by blunt dissection.</li>
	<li>Pull the eyelids apart using the thumb and index fingers, and place forceps under the eye globe. Close the forceps, and gently pull the eyeball off from the orbit.</li>
	<li>Place the eyeballs in 1x PBS. For each eyeball, remove the cornea from the eye globe by first making an incision using a 30 G needle below the limbus area.</li>
	<li>Cut around the limbus area using corneal micro scissors, with the incision as the starting point, and slowly separate the cornea and limbus from the globe.</li>
	<li>Clean the corneas gently using a fine paintbrush to remove the iris. Fix the corneas in 4% paraformaldehyde for 1 h.</li>
	<li>Wash the corneas three times for 20 min each in 1x PBS at room temperature (RT).</li>
	<li>Incubate in a blocking buffer (1x PBS supplemented with 0.1% Triton-X 100 and 5% bovine serum albumin [BSA]) for 1 h at RT.</li>
	<li>Transfer the corneas to an antibody solution containing primary antibodies. Prepare the antibody solution in 1x PBS supplemented with 1% BSA, 0.1% Triton-X 100, Dylight550-conjugated anti-CD31 mAb (1:100), and Alexa Fluor488-conjugated anti-LYVE-1 mAb (1:100).</li>
	<li>Incubate for 3 days at 4 &#176;C. Wash the corneas in 1x PBS three times for 20 min each.</li>
	<li>Stain the nuclei using Hoechst stain solution (1:1,000) for 5 min in the dark.</li>
	<li>Flatten the corneas with radial cuts, and mount them on a pre-cleaned glass slide using mounting medium and coverslips. Seal the coverslips with clear nail polish, and dry the slides overnight in the dark before analysis by confocal microscopy.</li>
	<li>Image the flat-mounted corneas using confocal microscopy by stitching individual Z-stack images; use a 10x objective, 488 nm and 561 nm lasers, and a resolution of 512 pixels x 512 pixels per slice on non-resonant galvano scanners.</li>
	<li>Quantify the density of CD31+ blood and LYVE-1+ lymph vessels using ImageJ software.</li>
	<li>To determine the vascular density, convert the confocal images to an 8-bit image.</li>
	<li>Choose Vascular Density from the Plugins.</li>
	<li>Choose the region of interest on the image, and click on OK. The measurements will open in a new data window.</li>
	<li>Copy the values to a spreadsheet file, and statistically compare the vascular density between the alkali burn and healthy eye groups using a Student&#39;s t-test. 
	NOTE: CD31, also called platelet endothelial cell adhesion molecule-1 (PECAM-1), is a cell adhesion molecule involved in angiogenesis and is highly expressed in the endothelial cells of early and mature blood vessels13. LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a cell surface marker on lymphatic endothelial cells and can be used as a lymphangiogenesis marker14.</li>
</ol>

Alkali Burn-Induced Corneal Neovascularization in Mice

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	<li>Ellenberg, D., 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=Novel+aspects+of+corneal+angiogenic+and+lymphangiogenic+privilege.">Novel aspects of corneal angiogenic and lymphangiogenic privilege.</a> Progress in Retinal and Eye Research. 29 (3), 208-248 (2010).</li><li>Azar, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+angiogenic+privilege:+Angiogenic+and+antiangiogenic+factors+in+corneal+avascularity,+vasculogenesis,+and+wound+healing+(an+American+Ophthalmological+Society+thesis).">Corneal angiogenic privilege: Angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).</a> Transactions of the American Ophthalmological Society. 104, 264-302 (2006).</li><li>Rolfsen, M. L., 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=Corneal+neovascularization:+A+review+of+the+molecular+biology+and+current+therapies.">Corneal neovascularization: A review of the molecular biology and current therapies.</a> Expert Review of Ophthalmology. 8 (2), 167-189 (2013).</li><li>Skobe, M., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+the+path+of+lymphatic+vessels.">Blocking the path of lymphatic vessels.</a> Nature Medicine. 15 (9), 993-994 (2009).</li><li>Lee, P., Wang, C. C., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+neovascularization:+An+epidemiologic+review.">Ocular neovascularization: An epidemiologic review.</a> Survey of Ophthalmology. 43 (3), 245-269 (1998).</li><li>Su, W., 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=Efficacious,+safe,+and+stable+inhibition+of+corneal+neovascularization+by+AAV-vectored+anti-VEGF+therapeutics.">Efficacious, safe, and stable inhibition of corneal neovascularization by AAV-vectored anti-VEGF therapeutics.</a> Molecular Therapy - Methods &amp; Clinical Development. 22, 107-121 (2021).</li><li>Lasagni Vitar, R. 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=Epidemiology+of+corneal+neovascularization+and+its+impact+on+visual+acuity+and+sensitivity:+A+14-year+retrospective+study.">Epidemiology of corneal neovascularization and its impact on visual acuity and sensitivity: A 14-year retrospective study.</a> Frontiers in Medicine. 8, 733538 (2021).</li><li>Said, D. G., Dua, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+burns+acid+or+alkali,+what's+the+difference.">Chemical burns acid or alkali, what's the difference.</a> Eye. 34, 1299-1300 (2020).</li><li>Muthukkaruppan, V. R., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+mouse+cornea.">Angiogenesis in the mouse cornea.</a> Science. 2 (4413), 1416-1418 (1979).</li><li>Kenyon, B. 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=A+model+of+angiogenesis+in+the+mouse+cornea.">A model of angiogenesis in the mouse cornea.</a> Investigative Ophthalmology &amp; Visual Science. 37 (8), 1625-1632 (1996).</li><li>Cursiefen, C., Maruyama, K., Jackson, D. G., Streilein, J. W., Kruse, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+angiogenesis+and+lymphangiogenesis+after+brief+corneal+inflammation.">Time course of angiogenesis and lymphangiogenesis after brief corneal inflammation.</a> Cornea. 25 (4), 443-447 (2006).</li><li>Yoeruek, 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=penetration+and+efficacy+of+topically+applied+bevacizumab:+Evaluation+of+eyedrops+in+corneal+neovascularization+after+chemical+burn.">penetration and efficacy of topically applied bevacizumab: Evaluation of eyedrops in corneal neovascularization after chemical burn.</a> Acta Ophthalmologica. 86 (3), 322-328 (2008).</li><li>DeLisser, H. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+uptake+and+degradation+of+the+lymphatic+endothelial+hyaluronan+receptor+LYVE-1.">Inflammation-induced uptake and degradation of the lymphatic endothelial hyaluronan receptor LYVE-1.</a> The Journal of Biological Chemistry. 282 (46), 33671-33680 (2007).</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>Chung, J. H., Fagerholm, P., Lindstr&#246;m, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+corneal+epithelium+following+a+standardized+alkali+wound.">The behaviour of corneal epithelium following a standardized alkali wound.</a> Acta Ophthalmologica. 65 (5), 529-537 (1987).</li><li>Chang, J. H., Gabison, E. E., Kato, T., Azar, D. T. <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> Current Opinion in Ophthalmology. 12 (4), 242-249 (2001).</li><li>Alves da Costa, T., Lang, J., Torres, R. M., Pelanda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+human+immune+system+mice+and+their+use+to+study+tolerance+and+autoimmunity.">The development of human immune system mice and their use to study tolerance and autoimmunity.</a> Journal of Translational Autoimmunity. 2, 100021 (2019).</li><li>vander Weyden, L., White, J. K., Adams, D. J., Logan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+genetics+toolkit:+Revealing+function+and+mechanism.">The mouse genetics toolkit: Revealing function and mechanism.</a> Genome Biology. 12 (6), 224 (2011).</li></ol>

The authors declare no conflicts of interest.

The cornea is an excellent tissue for studying angiogenesis and inflammation because it is accessible and avascular, meaning that neovascularization can be conveniently detected and documented. Corneal burn in rabbits, rats, and mice has been used to study corneal angiogenesis, inflammation and opacity, ulceration, perforation of the cornea, and fibrosis15,16,17. Moreover, the mouse model of corneal burn is valuable for testing various therapeutic strategies for angiogenesis and inflammation because mice have an immune system closely related to that of humans18. The availability of techniques to genetically manipulate the mouse genome also makes the species an excellent choice for this type of study19. The challenge in this research has been to develop a method of corneal burn that provides consistent, reproducible pathophysiology.
The alkali burn model is particularly useful for the pharmacological screening of drugs that modulate angiogenesis, inflammation, and fibrosis. The minimal requirements for reagents and resources, the simplicity of performing the alkali burn, and the benefits of the short duration of the protocol and the direct observation of the results make alkali burn on the mouse cornea a primary choice for pharmacological drug screening. However, a few precautions should be considered when performing this procedure to ensure consistency and reproducibility. Firstly, the filter paper must be placed at the center of the cornea to avoid burning other areas of the eye, especially the limbus, eyelids, and conjunctiva; secondly, the volume and concentration of NaOH should be appropriate to obtain consistent results from the alkali burn on the cornea. The filter must not be dripping wet but should have been soaked in the NaOH solution. The filter size and filter type and the normality and volume of the solution used in this method are optimized to avoid an overflow of NaOH. Using a different-sized filter paper or a higher or lower volume of NaOH would cause inconsistencies in the neovascularization. Thirdly, it is important to prevent the NaOH solution from absorbing CO2 in the room air by immediately tightening the tube cap of the solution after use and reducing the air/solution ratio. Care must be taken to use fresh alkali solutions to prevent inconsistencies in the neovascularization and to avoid corneal ulceration. Finally, extensive washing of all the NaOH solution from the eye and conjunctiva with saline is necessary to prevent further damage to the cornea and surrounding tissues of the eye. The thorough washing of the cornea and the adjacent tissues will also prevent symblepharon.
The protocol described here is an efficient and reliable method for studying the pathophysiology of corneal angiogenesis. This protocol can be further used to study corneal inflammation, fibrosis, and wound healing.

The cornea is an avascular tissue that maintains its transparency by establishing an angiogenic privilege1,2. Damage to the cornea can result in inflammation and the development of blood and lymph vessels, as well as fibrosis3. Corneal neovascularization (CoNV) leads to visual impairment and is the second leading cause of blindness worldwide4.&#160;CoNV affects about 1.4 million people in the United States per year5. CoNV can be induced by various factors, including chemical burns, infections, inflammation, and hypoxia3,6. Chemical burns are one of the most common ocular emergencies, and they account for about 13.2% of ocular trauma and require immediate assessment and treatment7. Chemical burns could be alkali or acid burns, but alkali burns cause more severe injury, as alkali penetrates deeper into the tissue8.
Mouse models of alkali burn are widely used to study CoNV and wound healing. Compared to the corneal pocket angiogenesis model9,10, alkali burn models are relatively straightforward to create and can also be used to study corneal inflammation, fibrosis, and epithelial proliferation. These models are also more closely related to clinical chemical burns than corneal suture models of angiogenesis11. With alkali burn, the otherwise avascular cornea develops blood vessels due to inflammation and an imbalance in anti-angiogenic and pro-angiogenic factors1,2. The drawbacks of corneal alkali burn models are the difficulties in controlling the area and severity of the alkali burn, the variation in corneal neovascularization, and unintentional burning of the adjacent tissues due to excess alkali solution. The purpose of this study is to describe a controlled corneal alkali burn model in mice using filter paper pre-soaked in sodium hydroxide solution. This model could be used to study angiogenic factors, anti-angiogenic therapeutic reagents, and other factors and reagents that could modulate inflammation and fibrosis.

<table><tbody><tr><td>0.9% Sodium Chloride Injection</td><td>Hospira</td><td>KL-7302</td><td></td></tr><tr><td>30 G Needle</td><td>McKesson</td><td>16-N3005</td><td></td></tr><tr><td>A1R Confocal</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Anti-CD31</td><td>Novus Biologicals</td><td>NB100-1642R</td><td></td></tr><tr><td>Anti-LYVE-1</td><td>Life technologies</td><td>53-0443-82</td><td></td></tr><tr><td>ASM Module</td><td>Heidelberg Engineering</td><td></td><td>Anterior segment objective</td></tr><tr><td>Biopsy Punch</td><td>McKesson</td><td>16-1309</td><td></td></tr><tr><td>BSA</td><td>Thermoscientific</td><td>9048-46-8</td><td></td></tr><tr><td>Coverslip</td><td>VWR International</td><td>22X22-1-601640G</td><td></td></tr><tr><td>Dissection Microscope</td><td>AmScope</td><td>SM-4TZ-30WY-10M3</td><td></td></tr><tr><td>Fluoromount-G</td><td>Electron Microscopy Sciences</td><td>17984-25</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>15000-02</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Forceps</td><td>Fisherbrand</td><td>12-000-157</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Roboz</td><td>RS-4905</td><td></td></tr><tr><td>Gonak Hypromellose&amp;nbsp;</td><td>Akorn</td><td>17478006412</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Sotware, Inc</td><td></td><td></td></tr><tr><td>Heating pad</td><td>K&amp;amp;H Pet Products</td><td>100213018</td><td></td></tr><tr><td>Hoescht</td><td>Life Technologies</td><td>62249</td><td></td></tr><tr><td>HRA + OCT Spectralis</td><td>Heidelberg Engineering</td><td></td><td></td></tr><tr><td>Insulin Syringe</td><td>Mckesson</td><td>102-SN310C31516P</td><td></td></tr><tr><td>Kimwipe</td><td>Kimberly Clark Professional</td><td>34155</td><td></td></tr><tr><td>Micro Cover Glass</td><td>VWR</td><td>48366-067</td><td></td></tr><tr><td>Microscissors</td><td>Roboz</td><td>RS-5110</td><td></td></tr><tr><td>Microscopic Slide</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>55881-500G</td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates and Dexamethasone&amp;nbsp;</td><td>Bausch &amp;amp; Lomb</td><td>24208-0795-35</td><td></td></tr><tr><td>Normal Serum</td><td>Jackson Immuno</td><td>008-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>158127-500G</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-027</td><td></td></tr><tr><td>Proparacaine HCl</td><td>Bausch &amp;amp; Lomb</td><td>24208073006</td><td></td></tr><tr><td>Saline</td><td>Henry Schein</td><td>1531042</td><td></td></tr><tr><td>SMZ125</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Syringe 10 mL</td><td>McKesson</td><td>16-S10C</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>TX1568-1</td><td></td></tr><tr><td>Whatmann Filter Paper</td><td>Cytiva</td><td>WHA1003323</td><td></td></tr></tbody></table>

a mouse model for corneal neovascularization by alkali burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus&#160;and adversely&#160;affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

This study describes a method to induce corneal angiogenesis in the mouse eye by alkali burn. The images obtained with the dissection microscope (Figure 1A,B) demonstrated significantly elevated neovascularization and opacity scores in the corneas in the alkali burn group (P &#60; 0.05; Figure 1C,D). The corneas that were collected on day 10 were further immunostained with anti-CD31 mAb for blood vessels and anti-LYVE-1 mAb for lymph vessels, respectively (Figure 2A-I). The alkali burn group showed significantly higher densities of blood and lymph vessels after 10 days (P &#60; 0.001 and P &#60; 0.05, respectively; Figure 2J,K). The thickness of the cornea, as imaged and quantified using OCT (Figure 3A,B), was observed to be significantly higher in the group with alkali burn (P &#60; 0.01; Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65289/65289fig01.jpg" /> 
Figure 1: Alkali burn-induced corneal neovascularization and opacity. (A,B)&#160;Corneal neovascularization sprouted from the limbus vessels toward the corneal center in the (B) alkali-burned mouse eye (A) but not healthy eye 10 days after the injury. (C,D) Quantification of the (C) corneal neovascularization and (D) opacity in panels A and B (&#177; SEM; t-test; *P &#60; 0.05; n = 3 eyes, 1 eye/mouse). The red arrows represent the limbus, and the yellow arrow indicates the sprouting new vessels. <a href="https://www.jove.com/files/ftp_upload/65289/65289fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65289/65289fig02.jpg" /> 
Figure 2: Corneal neovascularization and lymphangiogenesis caused by alkali burn. Immunohistochemistry revealed (A,D,G) blood and (B,E,H) lymph vessels using the anti-CD31 and anti-LYVE-1 mAbs, respectively. (A-C) The healthy mouse cornea. (D-I) The alkali-burned cornea 10 days post injury. (C,F,I) Superimposed images of CD31 and LYVE-1 signals. (G-I) Zoomed-in images for panels D-F. Scale bars = (A-F) 200 &#181;m and (G-I) 500 &#181;m. (J,K) Quantification of the blood and lymph vessel density in panels A-F, as indicated (&#177; SEM; t-test; *P &#60; 0.05; ***P &#60; 0.001; n = 3 eyes, 1 eye/mouse). <a href="https://www.jove.com/files/ftp_upload/65289/65289fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65289/65289fig03.jpg" /> 
Figure 3: Increase in corneal thickness caused by alkali burn. (A) An OCT image of a healthy mouse eye. (B) An OCT image of the mouse cornea 10 days post alkali burn. (C) Quantification of the corneal thickness in panels A and B, as measured at the center of the cornea (&#177; SEM; t-test; **P &#60; 0.01; n = 3 eyes, 1 eye/mouse). <a href="https://www.jove.com/files/ftp_upload/65289/65289fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies at JoVE.com

Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

A Mouse Model for Corneal Neovascularization by Alkali Burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...

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Biology

2.2K Views.  Baylor College of Medicine. This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization. 

Video: Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

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.

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 ...

Medicine

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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 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.

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

A Mouse Model for Corneal Neovascularization by Alkali Burn

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

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

A Mouse 5/6^th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model