Name: quantitative fundus autofluorescence for the evaluation of retinal diseases
Uploaded: 2016-03-11T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases at JoVE.com

We would like to thank our collaborators, Francois Delori, Tomas Burke, and Tobias Duncker.
Research Support: NIH/NEI R01 EY015520 (RTS, JPG), and unrestricted funds from Research to Prevent Blindness (RTB).

Ethics Statement: All patients enrolled in these studies were done so in accordance with approved institutional review board oversight at New York University School of Medicine.
1. Patient Selection and Initial Preparation for Imaging
Note: The following materials are required: 0.5% tropicamide ophthalmic solution, 2.5% phenylephrine ophthalmic solution, cSLO equipped with spectral domain optical coherence tomography (SD-OCT), and internal fluorescence reference.
...

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiological reviews. 85, 845-881 (2005).</li><li>Ach, T., 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=Quantitative+autofluorescence+and+cell+density+maps+of+the+human+retinal+pigment+epithelium.">Quantitative autofluorescence and cell density maps of the human retinal pigment epithelium.</a> Investigative ophthalmology &amp; visual science. 55, 4832-4841 (2014).</li><li>Ach, T., 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=Lipofuscin+redistribution+and+loss+accompanied+by+cytoskeletal+stress+in+retinal+pigment+epithelium+of+eyes+with+age-related+macular+degeneration.">Lipofuscin redistribution and loss accompanied by cytoskeletal stress in retinal pigment epithelium of eyes with age-related macular degeneration.</a> Investigative ophthalmology &amp; visual science. 56, 3242-3252 (2015).</li><li>Schmitz-Valckenberg, S., Jorzik, J., Unnebrink, K., Holz, F. G., Group, F. A. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+digital+scanning+laser+ophthalmoscopy+fundus+autofluorescence+images+of+geographic+atrophy+in+advanced+age-related+macular+degeneration.">Analysis of digital scanning laser ophthalmoscopy fundus autofluorescence images of geographic atrophy in advanced age-related macular degeneration.</a> Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 240, 73-78 (2002).</li><li>Weng, 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=Insights+into+the+function+of+Rim+protein+in+photoreceptors+and+etiology+of+Stargardt's+disease+from+the+phenotype+in+abcr+knockout+mice.">Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice.</a> Cell. 98, 13-23 (1999).</li><li>Holz, F. G., 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=Inhibition+of+lysosomal+degradative+functions+in+RPE+cells+by+a+retinoid+component+of+lipofuscin.">Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin.</a> Investigative ophthalmology &amp; visual science. 40, 737-743 (1999).</li><li>Sparrow, J. R., Nakanishi, K., Parish, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipofuscin+fluorophore+A2E+mediates+blue+light-induced+damage+to+retinal+pigmented+epithelial+cells.">The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.</a> Investigative ophthalmology &amp; visual science. 41, 1981-1989 (2000).</li><li>Smith, R. T., 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=Lipofuscin+and+autofluorescence+metrics+in+progressive+STGD.">Lipofuscin and autofluorescence metrics in progressive STGD.</a> Investigative ophthalmology &amp; visual science. 50, 3907-3914 (2009).</li><li>Delori, F., 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=Quantitative+measurements+of+autofluorescence+with+the+scanning+laser+ophthalmoscope.">Quantitative measurements of autofluorescence with the scanning laser ophthalmoscope.</a> Investigative ophthalmology &amp; visual science. 52, 9379-9390 (2011).</li><li>Burke, T. R., 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=Quantitative+fundus+autofluorescence+in+recessive+Stargardt+disease.">Quantitative fundus autofluorescence in recessive Stargardt disease.</a> Investigative ophthalmology &amp; visual science. 55, 2841-2852 (2014).</li><li>Duncker, T., 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=Quantitative+fundus+autofluorescence+and+optical+coherence+tomography+in+best+vitelliform+macular+dystrophy.">Quantitative fundus autofluorescence and optical coherence tomography in best vitelliform macular dystrophy.</a> Investigative ophthalmology &amp; visual science. 55, 1471-1482 (2014).</li><li>Duncker, T., 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=Quantitative+fundus+autofluorescence+distinguishes+ABCA4-associated+and+non-ABCA4-associated+bull's-eye+maculopathy.">Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull's-eye maculopathy.</a> Ophthalmology. 122, 345-355 (2015).</li><li>Greenberg, J. P., 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=Quantitative+fundus+autofluorescence+in+healthy+eyes.">Quantitative fundus autofluorescence in healthy eyes.</a> Investigative ophthalmology &amp; visual science. 54, 5684-5693 (2013).</li><li>Delori, F. C., Goger, D. G., Dorey, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+accumulation+and+spatial+distribution+of+lipofuscin+in+RPE+of+normal+subjects.">Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.</a> Investigative ophthalmology &amp; visual science. 42, 1855-1866 (2001).</li><li>Sparrow, J. R., 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=Quantitative+fundus+autofluorescence+in+mice:+correlation+with+HPLC+quantitation+of+RPE+lipofuscin+and+measurement+of+retina+outer+nuclear+layer+thickness.">Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.</a> Investigative ophthalmology &amp; visual science. 54, 2812-2820 (2013).</li><li>Delori, F. C., Webb, R. H., Sliney, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+permissible+exposures+for+ocular+safety+(ANSI+2000),+with+emphasis+on+ophthalmic+devices.">Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices.</a> Journal of the Optical Society of America. A, Optics, image science, and vision. 24, 1250-1265 (2007).</li></ol>

Abnormal RPE lipofuscin distribution, whether increased or decreased, is a sensitive marker of retinal disease and is generally associated with loss of sensory retina function. Here, we describe application of qAF for the evaluation of RPE lipofuscin. Incorporation of an internal fluorescent reference to correct for variable laser power and detector sensitivity9 alongside our standardized imaging technique allows for reliable quantitation of AF levels. It is our goal that this method will aid in the diagnosis ...

The retinal pigment epithelium (RPE) supports the function of the sensory retina through numerous processes 1. Age-related macular degeneration (AMD) is the most important cause of untreatable blindness in industrialized countries and is characterized by changes in the RPE, including loss of pigment, loss of function and atrophy. In AMD and in normal aging, the RPE accumulates fluorescent, lysosome-derived organelles containing phagocytosed photoreceptor fragments, referred to as lipofuscin granules. The accumulation of RPE lipofuscin has been thought to indicate oxidative dysfunction 1, but recent studies have shown that the RPE morphology remains normal in aged eyes with high lipofuscin levels 2. However, abnormal patterns of lipofuscin distribution, in particular loss of lipofuscin, are documented markers for AMD and AMD progression, both histologically and clinically 3,4
Defective processing of RPE lipofuscin has also been shown to occur in certain inherited retinal degenerations. Patients suffering from Stargardt disease (STGD) accumulate lipofuscin in the RPE at a young age, eventually developing vision loss similar to that seen in AMD 5. These findings suggested that lipofuscin accumulation may itself be toxic and drive RPE dysfunction 6,7. However, a detailed imaging study of subjects with STGD over time did not confirm that focal lipofuscin accumulation led to subsequent RPE loss 8. Hence, although lipofuscin abnormalities are markers for retinal degenerations, a role for direct toxicity of lipofuscin remains unproven.
The RPE is the most posterior cell layer of the retina, but generates the majority of fluorescent signal from the ocular fundus. Generation and detection of autofluorescence (AF) derived from the RPE can be performed using confocal scanning laser ophthalmoscopy (cSLO), which allows for visualization of the spatial distribution of fundus AF. Certain retinal degenerations demonstrate distinctive patterns of fundus AF, and AF imaging aids in the diagnosis and monitoring of these conditions. Although standard AF imaging is clinically important, quantitative AF (qAF) has become an important means of assessing RPE health. We and others have developed a standardized approach that can reliably determine qAF levels at specific retinal locations 9. qAF has potential applications in the diagnosis and monitoring of retinal conditions, and may also have utility in prognosis and risk stratification. In addition, the diagnostic capabilities of qAF have also been described for certain retinal disorders 10-12. Here, we provide step-wise details for performing our technique accompanied by a visual demonstration of its application in the evaluation of healthy and diseased eyes.

<table border="0" cellpadding="0" cellspacing="0" width="671">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Spectralis HRA + OCT</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.5% tropicamide ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2.5% phenylephrine ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Internal fluorescent reference</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IGOR Pro software</td>
			<td style="width:143px;">WaveMetrics</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative fundus autofluorescence for the evaluation of retinal diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

This technique was used to study qAF in both healthy 13 and disease states 10-12. In healthy eyes (Figure 1), AF emitted from the RPE is distributed relatively uniformly throughout the fundus (Figure 1A). Reduced intensity is seen in the central macular region due to blocking of light by macular pigment, and at the sides and corners of the image due to the optics of the eye and camera. Vessels appear dark and should be in clear focus...

Watch this Scientific Journal Video about Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases at JoVE.com

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

Research

JoVE Journal

Medicine

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Smartphone Fundus Photography

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...