This work was funded by the German Ophthalmologic Society (DOG) grant for doctoral students (MW) and the NIH/NEI 1R01EY027948 (TA).

Exploring Quantitative Autofluorescence in Age-Related Macular Degeneration

<ol>
	<li>Schmitz-Valckenberg, S., 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=Fundus+autofluorescence+imaging.">Fundus autofluorescence imaging.</a> Progress in Retinal and Eye Research. 81, 100893 (2021).</li><li>Bermond, K., 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=Autofluorescent+granules+of+the+human+retinal+pigment+epithelium:+phenotypes,+intracellular+distribution,+and+age-related+topography.">Autofluorescent granules of the human retinal pigment epithelium: phenotypes, intracellular distribution, and age-related topography.</a> Investigative Ophthalmology and Visual Science. 61 (5), 35 (2020).</li><li>Bermond, K., 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=Autofluorescent+organelles+within+the+retinal+pigment+epithelium+in+human+donor+eyes+with+and+without+age-related+macular+degeneration.">Autofluorescent organelles within the retinal pigment epithelium in human donor eyes with and without age-related macular degeneration.</a> Investigative Ophthalmology and Visual Science. 63 (1), 23 (2022).</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 and Visual Science. 52 (13), 9379-9390 (2011).</li><li>Fleckenstein, M., 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=Age-related+macular+degeneration.">Age-related macular degeneration.</a> Nature Reviews Disease Primers. 7 (1), 31 (2021).</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 and Visual Science. 54 (8), 5684-5693 (2013).</li><li>Sparrow, J. R., Boulton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE+lipofuscin+and+its+role+in+retinal+pathobiology.">RPE lipofuscin and its role in retinal pathobiology.</a> Experimental Eye Research. 80 (5), 595-606 (2005).</li><li>vonder Emde, L., 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=Natural+history+of+quantitative+autofluorescence+in+intermediate+age-related+macular+degeneration.">Natural history of quantitative autofluorescence in intermediate age-related macular degeneration.</a> Retina. 41 (4), 694-700 (2021).</li><li>Reiter, G. S., 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=Longitudinal+changes+in+quantitative+autofluorescence+during+progression+from+intermediate+to+late+age-related+macular+degeneration.">Longitudinal changes in quantitative autofluorescence during progression from intermediate to late age-related macular degeneration.</a> Retina. 41 (6), 1236-1241 (2021).</li><li>Gliem, M., 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+early+and+intermediate+age-related+macular+degeneration.">Quantitative fundus autofluorescence in early and intermediate age-related macular degeneration.</a> JAMA Ophthalmology. 134 (7), 817-824 (2016).</li><li>Hussain, R. M., Gregori, N. Z., Ciulla, T. A., Lam, B. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+quantitative+fundus+autofluorescence.">Lessons learned from quantitative fundus autofluorescence.</a> Progress in Retinal and Eye Research. 74, 100774 (2020).</li><li>Schmitz-Valckenberg, S., 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=Correlation+between+the+area+of+increased+autofluorescence+surrounding+geographic+atrophy+and+disease+progression+in+patients+with+AMD.">Correlation between the area of increased autofluorescence surrounding geographic atrophy and disease progression in patients with AMD.</a> Investigative Ophthalmology and Visual Science. 47 (6), 2648-2654 (2006).</li><li>Ach, T., Bermond, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+of+the+human+retinal+pigment+epithelium+in+normal+aging+and+in+age-related+macular+degeneration:+histology+and+clinical+correlation.">Autofluorescence of the human retinal pigment epithelium in normal aging and in age-related macular degeneration: histology and clinical correlation.</a> Klinische Monatsblatter Fur Augenheilkunde. 236 (5), 672-681 (2017).</li><li>Pollreisz, A., 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=Visualizing+melanosomes,+lipofuscin,+and+melanolipofuscin+in+human+retinal+pigment+epithelium+using+serial+block+face+scanning+electron+microscopy.">Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy.</a> Experimental Eye Research. 166, 131-139 (2018).</li><li>Bernstein, P. S., 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=meso-zeaxanthin:+The+basic+and+clinical+science+underlying+carotenoid-based+nutritional+interventions+against+ocular+disease.">meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.</a> Progress in Retinal and Eye Research. 50, 34-66 (2016).</li><li>G&#246;bel, A. P., Fleckenstein, M., Heeren, T. F. C., Holz, F. 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(109), 53577 (2016).</li><li>Pr&#246;bster, C., 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+the+developing+and+maturing+healthy+eye.">Quantitative fundus autofluorescence in the developing and maturing healthy eye.</a> Translational Vision Science and Technology. 10 (2), 15 (2021).</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+PRPH2/RDS-+and+ABCA4-associated+disease+exhibiting+phenotypic+overlap.">Quantitative fundus autofluorescence and optical coherence tomography in PRPH2/RDS- and ABCA4-associated disease exhibiting phenotypic overlap.</a> Investigative Ophthalmology and Visual Science. 56 (5), 3159-3170 (2015).</li></ol>

Leon von der Emde reports receiving payments from Heidelberg Engineering. Merten Mallwitz reports no financial disclosures. Kenneth R. Sloan also reports no financial disclosures. Frank G. Holz reports consulting/personal payments for Acucela, Alcon (C), Gyroscope Allergan Apellis, Bayer Bioeq/Formycon, CenterVue, Roche/Genentech, Geuder, Ivericbio, NightStarX, Novartis, Optos, Oxurion, Pixium Vision, Stealth BioTherapeutics, Zeiss, and GRADE Reading center. Thomas Ach reports consulting/personal payments for Bayer, Apellis, Roche, and Novartis.

This workflow provides a step-by-step guide to use open-source FIJI plug-in tools to determine and compare AF of AMD-specific lesions. The plug-ins provide easy-to-use templates that do not require any coding knowledge and can be applied by physicians without technical support27. To our knowledge, these tools are one of a kind for lesion-specific AF quantification.
QAF values naturally vary across the retina, with values being higher at the periphery and lower in the macula due to uneven lipofuscin and melanolipofuscin distribution within the retina, the low AF of vessels, and uneven macular pigment distribution. Due to the high variation of naturally occurring QAF levels in the retina, analyzing absolute QAF values of lesions directly is not a promising approach. For example, a hypoautofluorescent lesion in the periphery might still have higher absolute QAF values than physiologic fluorescence levels of the macula. The use of a StandardRetina and the use of z-scores to measure the fluorescence levels of drusen correct for this naturally occurring variance of QAF values.
A z-score is a numerical measurement of a region of interest value&#39;s relationship to the mean in the StandardRetina. It is calculated by subtracting the mean from an individual from the mean from the StandardRetina at the same location, and then dividing the result by the standard deviation. This standardization allows for the comparison of different QAF images, as the z-score indicates how many standard deviations a value differs from the mean. A positive z-score indicates that the value is above the mean, while a negative z-score indicates that it is below the mean.
It is important to note that there may be potential pitfalls that should be considered. While this method accounts for the varying amount of AF levels throughout the fundus, it may still not be the most accurate way to measure and compare an RPE&#39;s AF. Individuals have different levels and topography of macular luteal pigment, and lesions may affect the translucency of the overlying retina as well28,29. It is therefore plausible that the measured reduced AF in areas of SDDs (see representative results) is a consequence of shadowing effects rather than decreased fluorophores in the RPE30,31,32.
We are currently working on a workflow to account for retinal reflectivity, thickness, and quantified macular pigment (using green and blue AF) with linear mixed models. Additionally, so far, QAF uses an age-dependent correction factor to account for lenticular opacification that disregards interindividual differences in the lenticular opacification of participants of a similar age33. We are therefore currently working on a workflow for a personalized correction factor of lenticular autofluorescence and opacification. To reliably extract information of AF from small lesions, adequate test-retest reliability of QAF images is needed. To further differentiate those QAF images where more detailed analysis is viable, we are investigating &#34;QAF image reliability indices&#34; that can predict the test-retest reliability of QAF images. At the current stage, the prudent approach is to acquire duplicate images and investigate the retest reliability of lesion-specific AF.
The presented method of additionally analyzing the iso-hulls of lesions was technically difficult to implement, as iso-hulls of neighboring lesions merge. Areas of merged iso-hulls could be characterized distinctively depending on which lesion is considered. Our solution was to consider all lesions of one type as one lesion and to analyze their periphery as a joint iso-hull. This method, however, drastically reduces the ability to measure the iso-hulls of individual drusen and might be considered a further pitfall of this technique. More technically sophisticated methods to account for merged iso-hulls or suspended reporting of AF in areas of merged iso-hulls could facilitate the analysis of AF in the circumference of lesions in the future.
We used AMD as a model disease for this study. The workflow can be adapted to study lesions in other diseases as well. So far, QAF has been used in many chorioretinal diseases, including recessive Stargardt disease, Bestrophin-1 associated diseases, various forms of retinitis pigmentosa, acute zonal occult outer retinopathy, pseudoxanthoma elasticum, and others17,33,34,35,36,37. As this workflow uses open-source software, we encourage others to duplicate this work in determining lesion-specific AF and expand our knowledge of retinal disorders. In summary, we present a workflow to determine and compare AF levels of different retinal lesions throughout the macula. This workflow paves the way for more in-depth analysis of AF and could facilitate the development of new biomarkers in AMD and beyond.

Fundus autofluorescence (FAF) imaging provides a noninvasive mapping of naturally and pathologically occurring fluorophores of the ocular fundus1. The most common blue (488 nm excitation) autofluorescence (AF) excites lipofuscin and melanolipofuscin granules of the retinal pigment epithelium (RPE)2,3,4. The distribution and increase/decrease of granules play a central role in normal aging and various retinal diseases, including age-related macular degeneration (AMD)5.
A further development of FAF, quantitative fundus autofluorescence (QAF), now allows the accurate determination of topographically resolved retinal AF intensities4,6. By incorporating a reference into the optical pathway of the FAF imaging device, AF intensities can be compared between devices, time points, and subjects. This technique has resulted in a paradigm shift with regard to a presumed pathogenetic factor in AMD, which for a long time was speculated to be due to excessive lipofuscin accumulation in RPE cells7. Histologic and clinical quantification of AF, however, has revealed a decrease in AF in AMD (due to the redistribution and loss of autofluorescent lipofuscin and melanolipofuscin granules), instead of the proposed increase in AF8,9,10.
Monitoring AF has clinical implications. Von der Emde et al. and others have showed that AF is not only decreased but also further decreases in the course of AMD in high-risk, intermediate AMD eyes8,9. Additionally, histological studies suggest that most AMD-affected RPE cells show a characteristic behavior with granule aggregation and extrusion prior to RPE cell loss via subduction, sloughing, migration, or atrophy13,14,15,16. This further indicates that AF loss might be a trigger or a surrogate signal of impending disease progression.
QAF studies so far have only evaluated AF globally at the posterior pole-using prefabricated grid polar coordinate systems (e.g., QAF8/Delori Grid)17. Using prefabricated grids to measure AF results in multiple AF values on predetermined areas per eye of a subject. Investigating AF values in this way might miss local changes in areas with pathologically altered AF, for example, in AMD atop or close to drusen or subretinal drusenoid deposits (SDDs). Drusen, and to a higher degree SDDs, are associated with a high risk of developing late AMD and vision loss. Drusen in particular have a typical cycle of increasing in size over many years and may deteriorate rapidly prior to atrophy. It is conceivable that, for example, global AF decreases in AMD, but increases or is even further reduced in and around these specific disease-related focal lesions.
Different local AF patterns could also have prognostic relevance for disease progression. For example, autofluorescence levels might be used to assess whether drusen are increasing in size or are already in regression to atrophy. It has already been shown that altered AF perilesional patterns in geographic atrophy largely impact atrophy progression over time18. Additionally, local autofluorescence patterns could reveal further details into the health of the RPE. Oftentimes, the optical coherence tomography (OCT) shows hyperreflectance into the choriocapillaris, although the RPE layer appears intact. A multimodal approach combining local QAF values and OCT might help differentiate lesions with a high risk of RPE disruption and impending atrophy.
One reason spatially resolved analyses in studies have not been performed is because the most commonly used manufacturer&#39;s software does not provide a tool for these types of analysis. AF properties of different lesions dependent on the AMD disease stage could further explain the pathogenesis of AMD. Therefore, a tool to measure regional, lesion-specific AF would be desirable. To accurately compare lesions that are located throughout the retina, the workflow needs a way to account for varying degrees of AF in the human fundus19. Most centrally, AF is characteristically lower due to the shadowing effects of macular pigment and differing granule counts20,21.
AF reaches its peak at ~9&#176; (distance to the fovea in all directions) and decreases to a greater extent peripherally4. Therefore, if one were to compare absolute values of AF levels from soft drusen (located at the fovea and parafovea in low AF areas) and SDDs (located paracentrally in high AF areas), the results would not be comparable22. Inspired by the work by Pfau et al. and the concept of sensitivity loss (correcting sensitivity measured in AMD for the hill of vision [declining retinal sensitivity with distance to the fovea] of healthy controls) for fundus controlled perimetry, AF is compared to standardized AF values throughout the macula23,24. The results are reported as z-scores (numerical measurement of a region of interest value&#39;s relationship to the mean).
The purpose of this study is to evaluate the use of a new tool for measuring local QAF levels in different types of lesions in patients with AMD. This tool is designed to measure autofluorescence levels of lesions identified on OCT scans. This enables the assessment of local autofluorescence levels in lesions, such as soft drusen or SDDs, and allows for tracking of AF changes from lesions over time. The potential utility of this tool is to enable a new structural biomarker that estimates the health of the RPE and may have prognostic value for the investigated lesions.

<table><tbody><tr><td>BatchStandardRetina plugin</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr><tr><td>FIJI (Image J)</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Mark_Bscans_OCT plugin</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Microspft office</td><td>Microsoft</td><td>n.a.</td><td>n.a.</td></tr><tr><td>QAF_xml_reader plugin</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Register_OCT_2 plugin</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Spectralis</td><td>Heidelberg Engineering</td><td>n.a.</td><td>QAF extension</td></tr><tr><td>StandardRetina plugin</td><td>n.a.</td><td>n.a.</td><td>n.a.</td></tr></tbody></table>

a workflow to quantitatively determine age-related macular degeneration lesion-specific variations in fundus autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Viewing the output 
To adequately analyze and draw conclusions from the results, it is important to understand the output file of Mark_Bscans_OCT. The first three columns are labeled after the case ID, the laterality of the file, and the imaging modality that was chosen. The fourth column is referred to by mode and is labeled z-score. Note that as of writing this text, Mark BScans can only calculate all lesions in one go; the rows refer to iso-hulls, whose distances from the outer edge of the lesion are specified in the lower and upper columns of the spreadsheet. Iso-hulls measure AF in z-scores (in case of QAF) in a specified circumference surrounding the lesion. Note that the minimum value of a pixel in an iso-hull can be found in the columns labeled min, the columns labeled median, max, mean, and stdev, respectively contain the median, maximum, mean, and standard deviation of the mean of the pixel values in aniso-hull. The column n contains the total number of pixels in an iso-hull. Figure 1 shows a singular marked soft drusen of an 84-year-old male patient with intermediate age-related macular degeneration (iAMD).
Figure 2&#160;shows the left eye of a representative patient with SDDs marked with the QAF-Workflow tool (Figure 3). SDDs in this patient were associated with reduced AF (z-score = -0.4 &#177; 0.2). Similarly, the iso-hulls around the SDD demonstrated reduced AF (e.g., closest iso-hull = -0.3 &#177; 0.3) compared to the StandardRetina. A plausible explanation for this phenomenon might be shadowing effects (reduced translucency) of SDD lesions on the RPE. The use of SDDs was exemplary. The tool enables the assessment of local AF levels in other lesions, such as drusen, as well. Furthermore, the tool allows for tracking AF changes from lesions over time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65238/65238fig01.jpg" /> 
Figure 1: A singular marked soft drusen of an 84-year-old male patient with intermediate age-related macular degeneration (iAMD). (A) The QAF image of a left eye with the marked drusen. (B) Close-up of the drusen: brown center representing the marked drusen and colored bands representing the surrounding iso-hulls. The table below depicts the output file. QAF drusen values are compared to corresponding QAF values of the corresponding eccentricity from the StandardRetina. This results in z-scores that represent deviation from the mean of unaffected areas. The blue box shows from left to right: the case ID, laterality of the eye, modality used, and the desired output (in this case, z-scores). Columns within the orange box show the boundaries of the measured area in millimeters (lower = lower bound, upper = upper bound). The green box labels the columns showing the QAF measurements. From left to right, these contain the minimum, median, maximum, number of pixels, mean, and standard deviation of the mean. Each row represents an iso-hull, rows within the blue box represent values within the lesion, and rows within the purple box show the iso-hulls surrounding each lesion (from top to bottom with increasing distance to the lesion). Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65238/65238fig02.jpg" /> 
Figure 2: Marked SDDs in a QAF image of an 80-year-old female patient with early AMD. (A) SDDs can be seen in the QAF image. The same QAF image is shown with imprinted annotations of SDDs. (B) Around each marked lesion, the iso-hulls are depicted with color-coding (light green, dark green, and red). (C) A magnified version of the blue rectangle. The outer edge of each SDD is marked in blue. Abbreviations: QAF = quantitative autofluorescence; AMD = age-related macular degeneration; SDD = subretinal drusenoid deposit. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65238/65238fig03.jpg" /> 
Figure 3: Workflow to determine AF of lesions. This figure visualizes the software plug-ins needed to determine lesion-specific AF. (A) Image shows a color-coded QAF image that can be used to visualize the distribution of QAF values, but should not be used for further analysis. (B) A QAF image in the foreground, with the infrared image from the SD-OCT scan in the background is shown. This is supposed to visualize the alignment using vessel bifurcations. This can be done using the Register_OCT_2 plug-in. (C)&#160;A StandardRetina which is used to measure the z-score values of lesions. StandardRetinas can be created using StandardRetina/BatchStandardRetina.(D) A BScan with blue arrows pointing to SDDs, which are highlighted by yellow lines (note: lesions are always marked below the RPE independent of the location in the z direction) is depicted. (E) All marked lesions are seen imprinted on a QAF image (see Figure 1). The last two steps are done using the Mark_BScans_OCT plug-in. Abbreviations: AF = autofluorescence; QAF = quantitative autofluorescence; SDD = subretinal drusenoid deposit; IR = infrared; RPE = retinal pigment epithelium; SD-OCT = spectral domain optical coherence tomography. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow at JoVE.com

Author Spotlight: Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow 

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...

a-workflow-to-quantitatively-determine-age-related-macular

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  University of Bonn. This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Video: Author Spotlight: Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow 

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

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.

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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.

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 ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Author Spotlight: Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...