This work was supported by NIH grant 1R01EY022901, the Career Development Award from Research to Prevent Blindness (RPB), C.M.Reeves &#38; M.A. Reeves Foundation, E. Matilda Ziegler Foundation for the Blind, Knights Templar Eye Foundation, and an unrestricted grant to the Department of Ophthalmology at the University of Utah from RPB. &#160;We thank Balamurali Ambati for technical assistance on the Spectralis Multi-Modality Imaging System and Tao Zhang for discussions and comments on the manuscript.

1. Preparation of Instruments
<ol>
	<li>The procedure is performed in a procedure room in an animal facility.</li>
	<li>Wear facemasks, hair bonnets, surgical gowns, sterile foot-covers, and gloves before beginning the experiment.</li>
	<li>Heat water in a beaker to ~40 &#176;C on a hotplate.</li>
	<li>Place a sterile blue pad on top of a heating pad that will be used later to maintain the mouse&#39;s body temperature during imaging. Turn on the heating pad.</li>
	<li>Prepare the Imaging System:
	<ol>
		<li>Remove the ...

<ol>
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In this study, we demonstrated the use of ICGA to image choroidal lesions in HTRA1 transgenic mice. The characteristics of the early, middle, and late phases of ICGA in our mouse model match the time course well in human studies1. This is important to make better comparisons between human pathology and animal phenotypes, which are invaluable for research on pathophysiological mechanisms and treatment strategies of conditions related to the choroid such as AMD.
We first performed ICG...

Indocyanine green angiography (ICGA) is a diagnostic test to image problems related to blood vessels in the eye. The absorption spectrum of ICG ranges from 790-805 nm while the emission spectrum ranges from 770-880 nm with the peak emission at 835 nm&#160;1. This is different from the other popular dye, sodium fluorescein, whose spectrum falls in the visible range. The infrared spectrum enables ICG to penetrate through retinal pigment epithelium (RPE), serosanguineous fluid, and lipid exudates, all of which can easily block visualization by sodium-fluorescein based fluorescein angiography (FA). ICG is 98% protein-bound in the vasculature resulting in less extravasation, allowing enhanced imaging of choroidal vessels and choroidal lesions1,2. ICGA is almost the only choice to visualize choroidal vasculature, which is posterior to RPE. Figure 1 shows the comparison of ICGA and FA in imaging vasculature in mouse eyes. FA can be used to image the retinal vasculature well but not the choroidal vasculature. In contrast, ICGA can be used to image both retinal and choroidal vasculature. ICGA is performed with high-resolution digital imaging systems or scanning laser ophthalmoscopes (SLO) together with infrared-sensitive video cameras, which we will use in this study.
In the clinic, ICGA has been recommended in diagnosing a number of chorioretinal disorders involving the choroidal vasculature including Polypoidal Choroidal Vasculopathy (PCV), Retinal Angiomatous Proliferation (RAP), angioid streaks, vitelliform macular dystrophy, central serous chorioretinopathy, choroidal hemangioma, hemorrhaging retinal arteriolar macroaneurysms, choroidal tumors, and certain forms of posterior uveitis1,3. The combination of ICGA with FA and Optical Coherence Tomography (OCT) provide powerful tools for the clinicians in the diagnosis and management of exudative age-related macular degeneration (AMD)4-10. ICGA is especially useful for diagnosing conditions involving the choroid. In fact, ICGA is considered the gold standard for diagnosing PCV, a variant of exudative AMD11-13. PCV is characterized by a network of branching vessels with terminal polypoidal dilations in the choroidal vasculature11-13. PCV is frequently associated with recurrent serosanguineous detachments of the RPE and retina with leakage and bleeding from the polypoidal components11,14,15. We recently reported the generation of the first PCV animal model by transgenically expressing human HTRA1, a multi-functional serine protease, in mouse retinal pigment epithelium (RPE)16. We showed that increased HTRA1 induced characteristic features of PCV, e.g. polypoidal lesions.
Here we demonstrated the use of time-course ICGA by tail vein injection in AMD research using our HTRA1 mouse model. Our data suggest that IV-ICGA is superior to IP (or subcutaneous (SC))-ICGA that are currently used in the field17,18 for characterizing lesions in the choroid.
Statement on Animal Research&#160;
Animal experiments were conducted according to protocols approved by Institutional Animal Care and Use Committee (IACUC), and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

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	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

We performed ICGA time course in HTRA1 transgenic mice and control WT littermates, both of which are on the CD1 background. The albino CD1 background was selected to facilitate indocyanine green angiography (ICGA) imaging (see DISCUSSION). Some aneurism like dilations began to appear in the early phase in the HTRA1 mouse (Figure 2, a red arrow indicates the dilation at the tip of a vessel and a red circle indicates a cluster type polypoidal lesion). Choroidal vessels are clearly visible in both WT and HT...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

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13.5K Views.  University of Utah Health Sciences Center. Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Video: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography