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

<ol>
	<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. 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=Involvement+of+endothelial+PECAM-1/CD31+in+angiogenesis.">Involvement of endothelial PECAM-1/CD31 in angiogenesis.</a> The American Journal of Pathology. 151 (3), 671-677 (1997).</li><li>Johnson, L. A., Prevo, R., Clasper, S., Jackson, D. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Roboz</td>
			<td>RS-4905</td>
			<td></td>
		</tr>
		<tr>
			<td>Gonak Hypromellose&#160;</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&#38;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&#160;</td>
			<td>Bausch &#38; 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 &#38; 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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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 

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 ...

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, ...

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 ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...