Name: a mouse model of the cornea pocket assay for angiogenesis study
Uploaded: 2011-08-18T12:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study at JoVE.com

Our research is supported by the Intramural Research Program of the NIH, National Eye Institute.

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.
 <ol>
 <li>Make 10% (w/v) sucralfate solution with PBS. Store under room temperature.</li>
 <li...

<ol>
	<li>Cao, Y., Linden, P., Shima, D., Browne, F., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+angiogenic+activity+and+hypoxia+induction+of+heterodimers+of+placenta+growth+factor/vascular+endothelial+growth+factor.">In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.</a> The Journal of clinical investigation. 98, 2507-2511 (1996).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <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+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nature. 2, 2545-2550 (2007).</li><li>Poche, R. A., Saik, J. E., West, J. L., Dickinson, M. E. <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+cornea+as+a+transplantation+site+for+live+imaging+of+engineered+tissue+constructs.">The mouse cornea as a transplantation site for live imaging of engineered tissue constructs.</a> Cold Spring Harbor protocols. , 5416-54 (2010).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+is+dispensable+for+blood+vessel+growth+but+critical+for+their+survival,+and+VEGF-B+targeting+inhibits+pathological+angiogenesis.">VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. , 106-6152 (2009).</li><li>Tong, S., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+response+of+angiogenesis+to+basic+fibroblast+growth+factor+in+rat+corneal+pocket+assay:+I.+Experimental+characterizations.">Dose response of angiogenesis to basic fibroblast growth factor in rat corneal pocket assay: I. Experimental characterizations.</a> Microvascular research. 75, 10-15 (2008).</li><li>Muthukkaruppan, V., 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. 205, 1416-1418 (1979).</li><li>Ziche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+assay+for+angiogenesis.">Corneal assay for angiogenesis.</a> Methods in molecular medicine. 46, 131-142 (2001).</li><li>Ziche, M., Morbidelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+pocket+assay.">The corneal pocket assay.</a> Methods in molecular biology. 467, 319-329 (2009).</li></ol>

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...

<table border="1">
<tbody>
<tr>
	<th>Equipment</th>
	<th>Company</th>
	<th>Catalog number</th>
</tr>
<tr>
	<td>Dissecting microscope</td>
	<td>World Precision Instruments</td>
	<td>PZMIV-BS</td>
</tr>
<tr>
	<td>Von Graefe cataract knife (1.5x25mm blade, flat)</td>
	<td>Ambler Surgical</td>
	<td>3401E</td>
</tr>
<tr>
	<td>Dumont #5 forceps</td>
	<td>Fine Science Tools</td>
	<td>11252-50</td>
</tr>
<tr>
	<td>Slit lamp</td>
 <td>Zeiss</td>
	<td>30 SL-M</td>
</tr>
</tbody>
</table>
Table 1. Equipments

<table border="1">
<tbody>
<tr>
	<th>Reagent</th>
	<th>Company</th>
	<th>Catalog#</th>
 <th>Comments</th>
</tr>
<tr>
	<td>bFGF (basic fibroblast growth factor)</td>
	<td>PEPROTECH INC.</td>
	<td>100-18B</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sucralfate (Sucrose octasulfate&#x2013;aluminum complex)</td>
	<td>Sigma</td>
	<td>S0652</td>
 <td>10% in PBS</td>
</tr>
<tr>
	<td>poly-HEMA (Poly(2-hydroxyethyl methacrylate))</td>
	<td>Sigma</td>
	<td>P3932</td>
 <td>12% in Ethanol</td>
</tr>
</tbody>
</table>
Table 2. Reagents and materials

a mouse model of the cornea pocket assay for angiogenesis study

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.

Watch this Scientific Journal Video about A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study at JoVE.com

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

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.

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 ...

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