Name: a mouse model for laser-induced choroidal neovascularization
Uploaded: 2015-12-27T14:00:00
Description: Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

All animals are treated in accordance with the Guide of the Care and Use of Laboratory Animals 2013 Edition, the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and as approved by the Institutional Animal Care and Use Committee for Northwestern University.
Note: The following procedure can be done entirely with one operator; however, it is much more efficiently conducted with two operators with the tasks split acco...

<ol>
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K., van Rooijen, N., Ambati, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Depletion+Inhibits+Experimental+Choroidal+Neovascularization.">Macrophage Depletion Inhibits Experimental Choroidal Neovascularization.</a> IOVS. 44, 3578-3585 (2003).</li><li>Espinosa Heidmann, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+Is+Major+Stimulator+in+Model+of+Choroidal+Neovascularization.">VEGF Is Major Stimulator in Model of Choroidal Neovascularization.</a> IOVS. 41, 3158-3164 (2000).</li><li>Reich, S. J., 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=Small+interfering+RNA+(siRNA)+targeting+VEGF+effectively+inhibits+ocular+neovascularization+in+a+mouse+model.">Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.</a> Molr Vis. 9, 210-216 (2003).</li><li>Baffi, J., Byrnes, G., Chan, C. C., Csaky, K. 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There are multiple factors that can affect laser delivery and resultant CNV lesion development after successful laser-induction. These factors should be controlled for and standardized in order to have the most reliable results. The most pertinent of these factors are mouse selection (genotype, age, and sex), anesthetic selection, and laser settings.
The specific mouse model used can have a significant effect on the course of CNV development. The most widely used genotype is the C57BL/6 mouse....

Age-related macular degeneration (AMD) is one of the leading causes of blindness in individuals over the age of 501-3. AMD can be classified into two forms: atrophic (&#8220;dry&#8221;) AMD and neovascular (&#8220;wet&#8221;) AMD. The former is characterized by geographic atrophy of the retinal pigment epithelium (RPE), choriocapillaris, and photoreceptors, while the latter is characterized by the invasion of abnormal vessels from the choroid into the outer retinal layers causing leakage, hemorrhage, and fibrosis, and ultimately leading to blindness1,2. Of the two forms, neovascular AMD accounts for the majority of vision loss1. Fortunately, this form has numerous effective pharmacological management options, whereas its atrophic counterpart currently has no proven medical treatments3. Moreover, because the neovascular form has been easily re-capitulated in an animal model, it has been more widely accessible to basic AMD research exploring the underlying pathological mechanisms in order to develop novel therapies4.
The first animal model of experimental choroidal neovascularization (CNV) was developed by Ryan et al. in non-human primates5. This model induced rupture of Bruch&#8217;s membrane via laser photocoagulation, which caused a local inflammatory response resulting in angiogenesis similar to that seen in neovascular AMD. The histopathological progression of angiogenesis post-laser induction was found to mimic neovascular AMD, which confirmed the model&#8217;s validity6. Non-human primates offer the most similar anatomy to humans, but unfortunately, are expensive to maintain, cannot be easily genetically manipulated, and have a slow time course of disease progression7. Contrastingly, rodent models are much more cost-effective to maintain, can be genetically manipulated with relative ease, and have a much faster course of disease progression (experiments can be conducted on a time scale of weeks versus months). These experiments should only be conducted in pigmented rodents as it is very difficult to visualize in albino animals.
The mouse laser-induced CNV model, first developed by the Campochiaro group in the late 90&#8217;s10, has grown to be the dominant animal model in the majority of recent studies11-16. Due to the complex and still unclear pathogenesis of CNV, the laser model has been applied in all aspects of wet AMD research ranging from studying the molecular mechanisms driving angiogenesis to evaluating new treatment modalities for future human use. For example, Sakurai et al. and Espinosa-Heidmann et al. used the laser model to investigate the effect of macrophages on the development of CNV using transgenic mice and pharmacological depletion treatments15, 16. Giani et al. and Hoerster et al. used optical-coherence tomography (OCT) to image the laser-induced CNV in an effort to characterize the progression of CNV and compare the histopathologic findings to the findings seen on OCT imaging12,17. Finally, studies involving intravitreal injection of anti-angiogenic agents have been used as pre-requisites for human trials and were vital in developing the first generation of anti-VEGF agents used in management of neovascular AMD today10,18,19.
Alternative models for experimental CNV utilize surgical methods to induce CNV. This procedure involves injecting pro-angiogenic substances (e.g. recombinant viral vectors overexpressing VEGF, subretinal injection of RPE cells and/or polystyrene beads) to mimic the increased VEGF expression seen in neovascular AMD, with the goal of causing angiogenesis8,20. However, this method yields a drastically lower incidence of neovascularization; these studies showed that CNV in C57/BL6 mice occurs in 31% of injections versus the ~70% success rate seen in the laser photocoagulation method in the same strain of mice8,14. For these reasons, and given the advantages of using rodents versus non-human primates, the mouse model of laser-induced CNV has become the standard animal model of CNV for most neovascular AMD study experiments8.
The mouse eye is a miniscule, delicate tissue to work with. Maneuvering of the eye to visualize the retina is difficult and requires much practice until mastery is achieved. This task is complicated by the fact that it must be learned with the dominant and non-dominant hand. Furthermore, after the fine movements required to visualize the retina have been learned, the coordination between both hands and the foot pedal operating the laser are important. In this paper, we sought to distill the challenges of learning all of the physical manipulations involved in the laser-induced CNV procedure into a guide that would help operators achieve rapid success with this model.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

a mouse model for laser-induced choroidal neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Quantification of CNV lesions can be performed through analysis of flat-mounted choroids using immunofluorescence staining to label the CNV vessels. The two most frequently employed methods of tissue preparation are FITC-dextran labeling, done via perfusion immediately before animal sacrifice, or post-mortem immuno-staining with an endothelial cell marker. Both of these methods have been described previously in detail13,14,21; Figures 1 and 2 show examples of each, respectivel...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

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