Published: June 30th, 2023
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.
Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus and adversely 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.
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. 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.
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
2. Preparation of the 4% paraformaldehyde (PFA) solution
3. Preparing the ketamine/xylazine cocktail
4. Alkali burn on the mouse cornea
5. Examination and assessment of neovascularization and opacity
6. Optical coherence tomography (OCT) imaging
7. Immunostaining for CoNV on flat-mount corneas
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 < 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 < 0.001 and P < 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 < 0.01; Figure 3C).
Figure 1: Alkali burn-induced corneal neovascularization and opacity. (A,B) 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 (± SEM; t-test; *P < 0.05; n = 3 eyes, 1 eye/mouse). The red arrows represent the limbus, and the yellow arrow indicates the sprouting new vessels. Please click here to view a larger version of this figure.
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 µm and (G-I) 500 µm. (J,K) Quantification of the blood and lymph vessel density in panels A-F, as indicated (± SEM; t-test; *P < 0.05; ***P < 0.001; n = 3 eyes, 1 eye/mouse). Please click here to view a larger version of this figure.
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 (± SEM; t-test; **P < 0.01; n = 3 eyes, 1 eye/mouse). Please click here to view a larger version of this figure.
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 authors declare no conflicts of interest.
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.
|0.9% Sodium Chloride Injection
|30 G Needle
|Anterior segment objective
|Electron Microscopy Sciences
|Fine Science Tools
|Fine Science Tools
|GraphPad Prism 9
|GraphPad Sotware, Inc
|K&H Pet Products
|HRA + OCT Spectralis
|Kimberly Clark Professional
|Micro Cover Glass
|Neomycin and Polymyxin B Sulfates and Dexamethasone
|Bausch & Lomb
|Bausch & Lomb
|Syringe 10 mL
|Whatmann Filter Paper
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