The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.
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Method Article
A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study
In This Article
Summary
Abstract
A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.
Protocol
All equipments and reagents used are sterile. The protocol was approved by the Animal Care and Use Committee (ACUC) at the NEI/NIH (animal study protocol NEI-553), and was performed according to the NIH guidelines and regulations.
1. Producing the angiogenic factor-containing pellets
In this part, the pellets containing the angiogenic factor of interest are prepared.
- Make 10% (w/v) sucralfate solution with PBS. Store under room temperature.
- Make 12% (w/v) poly-HEMA with absolute ethanol. Store under room temperature.
- Make 1 μg/μl bFGF solution. Store in -80 °C.
- To mak....
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Discussion
The mouse cornea pocket assay model was first described in 1979 by Muthukkaruppan VR and Auerbach R 6. Detailed protocols have been published by different laboratories 2,3,7,8. Our laboratory has modified and used this method in our studies for many years 4. The mouse cornea pocket assay is a relatively simple model with great reproducibility. Another advantage of this model is that since the cornea normally lacks blood vessels, there is minimum amount of background in this assay. Therefo.......
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Disclosures
No conflicts of interest declared.
Acknowledgements
Our research is supported by the Intramural Research Program of the NIH, National Eye Institute.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Dissecting microscope | World Precision Instruments, Inc. | PZMIV-BS | |
| Von Graefe cataract knife (1.5x25mm blade, flat) | Ambler Surgical | 3401E | |
| Dumont #5 forceps | Fine Science Tools | 11252-50 | |
| Slit lamp | Carl Zeiss, Inc. | 30 SL-M | |
| Table 1. Equipments | |||
| bFGF (basic fibroblast growth factor) | PeproTech Inc | 100-18B | |
| Sucralfate (Sucrose octasulfate—aluminum complex) | Sigma-Aldrich | S0652 | 10% in PBS |
| poly-HEMA (Poly(2-hydroxyethyl methacrylate)) | Sigma-Aldrich | P3932 | 12% in Ethanol |
| Table 2. Reagents and materials |
References
- Cao, Y., Linden, P., Shima, D., Browne, F., Folkman, J. In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. The Journal of clinical investigation. 98, 2507-2511 (1996).
- Rogers, M. S., Birsner, A. E., D'Amato, R. J.
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