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1National Eye Institute
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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.
Tang, Z., Zhang, F., Li, Y., Arjunan, P., Kumar, A., Lee, C., et al. A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study. J. Vis. Exp. (54), e3077, doi:10.3791/3077 (2011).
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.
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.
2. Performing the mouse cornea pocket assay
In this part, we will demonstrate how to make a pocket in the mouse cornea, and how to insert a pellet into the pocket. The angiogenic factor contained in the pellet will be released gradually to the surrounding areas of the cornea (Fig. 1). In this demonstration, we use 8-week female C57/Bl6 mice.
3. Representative results
Figure 1. A. Normal cornea with H&E staining. No blood vessel exists in a normal cornea. B. Make a pocket underneath the epithelium layer of the cornea. C. Embed a pellet into the pocket. D. The angiogenic factor is released from the pellet, and new blood vessels grow from the normal limbal vessels.
Figure 2. Representative result five days after implantation of the pellet. A. Cornea with a pellet containing the vehicle only. No new blood vessel formation in the cornea. Only the pre-existing normal limbal vessels are visible. B. Cornea with a pellet containing bFGF. Robust new blood vessels (dot-lined) grew from the pre-existing normal limbal vessels.
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. Therefore, this model is commonly used by the field to study the angiogenic effect of different factors and molecules. Compared with the eyes of some larger animals, such as rats and rabbits, the mouse eyes are smaller and difficult to handle. Special care is therefore required when performing this model, especially during the processes of making the pocket in the cornea and inserting the pellet. The knife must be handled in a precisely controlled manner so that it will not puncture the cornea.
No conflicts of interest declared.
Our research is supported by the Intramural Research Program of the NIH, National Eye Institute.
|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|
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