Ophthalmic Surgery, Lasers and Imaging Retina

Clinical Science Imaging 

Noninvasive Imaging of Mitochondrial Dysfunction in Dry Age-Related Macular Degeneration

Matthew G. Field, MS; Grant M. Comer, MD, MS; Takahiro Kawaji, MD, PhD; Howard R. Petty, PhD; Victor M. Elner, MD, PhD

Abstract

BACKGROUND AND OBJECTIVE:

Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of age-related macular degeneration (AMD). Because increased flavoprotein fluorescence (FPF) is indicative of mitochondrial dysfunction, the authors attempted to detect mitochondrial dysfunction in eyes with AMD using FPF.

PATIENTS AND METHODS:

Six nonexudative eyes with AMD, including three with geographic atrophy (GA), and age-matched control eyes were imaged with a FPF device. Qualitative and quantitative analyses were conducted on the FPF images.

RESULTS:

Five eyes with AMD, including all three eyes with GA, showed qualitative and/or quantitative FPF heterogeneity that was not present in control eyes. Mean FPF average intensities of eyes with AMD with (P = .044) and without (P = .00060) GA were significantly greater than those of control eyes. The standard deviations of FPF images were greater in eyes with AMD (P = .020).

CONCLUSION:

In this small cluster of patients with AMD, retinal FPF is increased, suggesting elevated mitochondrial dysfunction. FPF heterogeneity indicates that an increased variability in mitochondrial dysfunction seems to be present in eyes with advanced disease.

From the Departments of Ophthalmology and Visual Sciences (MGF, GMC, TK, HRP, VME), Microbiology and Immunology (HRP), and Pathology (VME), University of Michigan, Ann Arbor, Michigan.

Supported by a grant from the Michigan Universities Commercialization Initiative and research funding from OcuSciences, Inc.

Drs. Elner and Petty have a financial interest in the presented material by having founded OcuSciences, Inc., to commercialize this technology. The remaining authors have no financial or proprietary interest in the materials presented herein.

Address correspondence to Victor M. Elner, MD, PhD, University of Michigan, Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105. E-mail: velner@umich.edu

Received: December 30, 2011
Accepted: April 27, 2012
Posted Online: July 19, 2012

Abstract

BACKGROUND AND OBJECTIVE:

Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of age-related macular degeneration (AMD). Because increased flavoprotein fluorescence (FPF) is indicative of mitochondrial dysfunction, the authors attempted to detect mitochondrial dysfunction in eyes with AMD using FPF.

PATIENTS AND METHODS:

Six nonexudative eyes with AMD, including three with geographic atrophy (GA), and age-matched control eyes were imaged with a FPF device. Qualitative and quantitative analyses were conducted on the FPF images.

RESULTS:

Five eyes with AMD, including all three eyes with GA, showed qualitative and/or quantitative FPF heterogeneity that was not present in control eyes. Mean FPF average intensities of eyes with AMD with (P = .044) and without (P = .00060) GA were significantly greater than those of control eyes. The standard deviations of FPF images were greater in eyes with AMD (P = .020).

CONCLUSION:

In this small cluster of patients with AMD, retinal FPF is increased, suggesting elevated mitochondrial dysfunction. FPF heterogeneity indicates that an increased variability in mitochondrial dysfunction seems to be present in eyes with advanced disease.

From the Departments of Ophthalmology and Visual Sciences (MGF, GMC, TK, HRP, VME), Microbiology and Immunology (HRP), and Pathology (VME), University of Michigan, Ann Arbor, Michigan.

Supported by a grant from the Michigan Universities Commercialization Initiative and research funding from OcuSciences, Inc.

Drs. Elner and Petty have a financial interest in the presented material by having founded OcuSciences, Inc., to commercialize this technology. The remaining authors have no financial or proprietary interest in the materials presented herein.

Address correspondence to Victor M. Elner, MD, PhD, University of Michigan, Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105. E-mail: velner@umich.edu

Received: December 30, 2011
Accepted: April 27, 2012
Posted Online: July 19, 2012

Introduction

Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness in patients older than 60 years. The prevalence of AMD in the United States according to the 2005–2008 National Health and Nutrition Examination Survey is approximately 6.5% of the population 40 years and older, or 8 million individuals.1 Within this cohort, 900,000 are estimated to have late stage AMD, which includes both the dry form (geographic atrophy [GA]) and the wet form (that complicated by choroidal neovascularization). Specifically, 550,000 adults 40 years and older are estimated to have GA,1 which is defined as any sharply delineated area measuring at least 0.175 mm in diameter with apparent absence of retinal pigment epithelium (RPE) and more visible choroidal vessels than in surrounding areas.1,2 The cause of GA is unclear, but initial cell loss is thought to occur in the RPE, in association with photoreceptor cell loss and choriocapillaris atrophy.3–5

Oxidative damage has been implicated as a contributor to the pathogenesis of early and late stage AMD, including GA.6–10 This hypothesis is supported by studies showing that daily antioxidant and zinc treatment improves visual acuity in patients with dry AMD and slows the rate of disease progression.6,7 Additionally, a proteomic study of drusen deposits in early AMD shows increased levels of protein adducts, including carboxyethylpyrrole, that are likely to be caused by reactive lipid and carbohydrate oxidation products,8 further suggesting that oxidative stress may play a part in the pathogenesis of AMD. Moreover, histopathologic studies in eyes with advanced AMD show even more pronounced retinal oxidative damage, with increased levels of biomarkers for lipid peroxidation, protein oxidation, and oxidative DNA damage.9,10

In cultured human RPE cells, oxidative damage has been shown to cause mitochondrial DNA mutations that compromise mitochondrial redox function and promote apoptosis.11,12 Several studies have shown mitochondrial DNA alterations and mutations in eyes with AMD,13–16 indicating that high levels of mitochondrial DNA damage are related to AMD.16 Mitochondrial proteomic analysis of the RPE in eyes with AMD show significant changes in expression of proteins implicated in mitochondrial functions, including DNA translation, protein folding, nuclear-encoded protein importation, ATP-synthase activity, and regulation of apoptosis.17,18 The number of RPE mitochondria and mitochondrial cristae, as well as total mitochondrial area, are decreased significantly in eyes with AMD compared to eyes of age-matched controls.19 Studies on eyes with advanced AMD show increased apoptosis in the inner choroid, RPE, and outer and inner nuclear layers.20

Oxidative stress impairs enzymatic complexes of the electron transport chain and reduces mitochondrial membrane potential (ΔΨm) before the occurrence of apoptosis.21 During oxidative stress and mitochondrial dysfunction, flavoproteins linked to these enzymatic complexes become oxidized and, when excited by blue light, emit green flavoprotein fluorescence (FPF).22,23 We have shown that FPF detects mitochondrial dysfunction in several retinal diseases.24–27 Thus, because oxidative stress and mitochondrial alterations have been implicated in AMD pathogenesis, we have verified the utility of FPF imaging to detect specific macular alterations in eyes with AMD.

Patients and Methods

FPF Alpha-Prototype Imaging

Using concepts employed for FPF imaging in previous human studies,24–27 we produced a proprietary FPF device containing a novel optical pathway that enriches the retinal signal while significantly reducing lenticular contributions, providing a visible FPF image of the retina, with blood vessels visible as hypofluorescent structures. Using customized software, hardware, and electronics, the FPF device (Fig. 1A) captures a 30° infrared (860 nm; EXS8810-2411 Superluminescent Light-Emitting Diode (LED); Exalos AG, Schlieren, Switzerland) fundus image (Fig. 1B) with a line-scanning laser ophthalmoscope (Physical Sciences Inc., Andover, MA), a pupil image ensuring proper light beam alignment into the eye with a USB-linked camera (Blue Fox; Matrix Vision GmbH, Oppenweiler, Germany), and a specialized 15° metabolic FPF image (Fig. 1C) with an electron-multiplying charge-coupled device camera (Photometrics 512B; Roper Scientific, Tucson, AZ). The device contains customized 467-nm excitation and 535-nm emission filters (Omega Optical, Brattleboro, VT) and a blue LED light source (Luxeon K2; Philips LumiLEDs Lighting Company, San Jose, CA). The yellow circle in the center of the captured infrared fundus image (Fig. 1B) represents the exact location of where the FPF metabolic image was taken (yellow overlay, Fig. 1C). These two images are par focal to each other, are both captured during the same acquisition, and can be overlaid on top of each other for image analysis. A set of optical masks and a customized dichroic filter is used to couple blue/green light into and out of the entrance and exit apertures imaged into the eye’s pupil and these apertures are physically separated by a grating to minimize contamination of the retinal FPF signal by lenticular fluorescence and light scatter.

Flavoprotein fluorescence (FPF) clinical instrument and image acquisition. Imaging with the FPF device; the optical head unit is 6 × 6 × 10 inches (1A). Infrared fundus image with FPF acquisition area highlighted in yellow (1B) and retinal FPF image (1C) of a normal 25-year-old human left eye.

Figure 1. Flavoprotein fluorescence (FPF) clinical instrument and image acquisition. Imaging with the FPF device; the optical head unit is 6 × 6 × 10 inches (1A). Infrared fundus image with FPF acquisition area highlighted in yellow (1B) and retinal FPF image (1C) of a normal 25-year-old human left eye.

An experienced ophthalmic engineering firm conducted light safety analyses on the FPF prototype and determined that all of the imaging beams are more than 100 times below the International Electrotechnical Commission’s maximum permissible energy levels28 and that both the laser and LED components of our device can be classified under the safest laser/LED category, type I. In fact, the FPF imaging beam puts significantly less energy into a patient’s eye than a light source from a standard fundus camera.

Patient Recruitment and FPF Imaging

After obtaining Institutional Review Board approval, an observational clinical investigational study was conducted at the University of Michigan to take FPF retinal images of patients with dry AMD with and without GA and age-matched controls. A retinal specialist (GMC) identified six appropriate cases from the patient population at the University of Michigan W. K. Kellogg Eye Center who met inclusion criteria and did not meet any exclusion criteria (Table 1). In total, 21 eyes with AMD of 15 patients were examined. Fifteen eyes were excluded for the following reasons: previous therapy for AMD other than taking vitamins (6), severe cataracts (3), history of diabetes mellitus (3), and outside of desired age range (3). Spouses and patients between the ages of 65 and 85 years who did not meet any exclusion criteria (Table 1) and had no evidence for or history of AMD were enrolled as age-matched controls. Ten eyes of five age-matched control patients were examined. Four eyes were excluded because of severe cataracts.

Inclusion and Exclusion Criteria

Table 1: Inclusion and Exclusion Criteria

Each enrolled individual underwent a complete ophthalmic examination by a retinal specialist to evaluate the areas of macular atrophy and to categorize the severity of AMD based on the Age-Related Eye Disease Study criterion.29 The six eyes with AMD were from five different patients; three eyes had moderate AMD and three had advanced AMD with GA. All control patients had normal funduscopic examinations. Of the six control eyes that met inclusion criteria and did not meet exclusion criteria, three eyes, each from a different patient, were randomly selected for inclusion in this study. Participants were recruited between February and June 2010 and were between 71 and 84 years of age. Oral and written consent was obtained from each participant before enrollment.

After pupillary dilation, multiple retinal FPF images and corresponding infrared fundus images were obtained from the macula of each eye. The depth of focus of the instrument resulted in capture of FPF from all retinal layers. FPF images, stored as 512 × 512 pixel files, were analyzed with specialized computer algorithms. An ophthalmologist (TK) without any prior knowledge of patients’ AMD status performed FPF imaging. Each subject also had color fundus images taken of each eye with a Zeiss FF450 Plus fundus camera (Carl Zeiss Corporation, Oberkochen, Germany). All images and analyses were independently read and interpreted by two retinal specialists.

FPF Image Analysis

A 400 × 160 pixel rectangular area of analysis was selected in the central region of the captured 512 × 512 pixel FPF image. This central region selection eliminated artifact contributions in the periphery of the FPF image caused by incident light (467-nm) scatter and diffraction of FPF due to the horizontal grating in the optical pathway. The horizontal grating separates the incident and returning light pathways to greatly reduce the possible lenticular artifacts. The overall FPF average intensity was calculated from all pixels in the central region selection delineated by a black rectangular overlay (Figs. 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, and 10A). The retinal FPF average intensity of control eyes was corrected to account for the mean difference in two-dimensional mitochondrial area between eyes with AMD and control eyes,19 because eyes with AMD have reduced mitochondrial area and thus a reduced maximum possible FPF when compared to that of the control eyes.

Flavoprotein fluorescence (FPF) of normal eyes. Color fundus photographs with the FPF analysis area highlighted in black (2A, 3A, 4A), corresponding FPF images (2B, 3B, 4B), and FPF images after anisotropic diffusion filtration (2C, 3C, 4C) of 73- (2A, 2B, 2C), 79- (3A, 3B, 3C), and 84- (4A, 4B, 4C) year-old eyes.

Figures 2, 3, and 4. Flavoprotein fluorescence (FPF) of normal eyes. Color fundus photographs with the FPF analysis area highlighted in black (2A, 3A, 4A), corresponding FPF images (2B, 3B, 4B), and FPF images after anisotropic diffusion filtration (2C, 3C, 4C) of 73- (2A, 2B, 2C), 79- (3A, 3B, 3C), and 84- (4A, 4B, 4C) year-old eyes.

Flavoprotein fluorescence (FPF) of moderate dry (nonexudative) eyes with age-related macular degeneration. Color fundus photographs with the selected FPF analysis area highlighted in black (5A, 6A, 7A), corresponding FPF images (5B, 6B, 7B), and FPF images after anisotropic diffusion filtration (5C, 6C, 7C) of 74- (5A, 5B, 5C), 74- (6A, 6B, 6C), and 78- (7A, 7B, 7C) year-old eyes with moderate dry age-related macular degeneration.

Figures 5, 6, and 7. Flavoprotein fluorescence (FPF) of moderate dry (nonexudative) eyes with age-related macular degeneration. Color fundus photographs with the selected FPF analysis area highlighted in black (5A, 6A, 7A), corresponding FPF images (5B, 6B, 7B), and FPF images after anisotropic diffusion filtration (5C, 6C, 7C) of 74- (5A, 5B, 5C), 74- (6A, 6B, 6C), and 78- (7A, 7B, 7C) year-old eyes with moderate dry age-related macular degeneration.

Flavoprotein fluorescence (FPF) of advanced dry (nonexudative) eyes with age-related macular degeneration with geographic atrophy. Color fundus photographs with the selected FPF analysis area highlighted in black (8A, 9A, 10A), corresponding FPF images (8B, 9B, 10B), and FPF images after anisotropic diffusion filtration (8C, 9C, 10C) of 71- (8A, 8B, 8C), 79- (9A, 9B, 9C), and 83- (10A, 10B, 10C) year-old eyes with advanced dry age-related macular degeneration and geographic atrophy.

Figures 8, 9, and 10. Flavoprotein fluorescence (FPF) of advanced dry (nonexudative) eyes with age-related macular degeneration with geographic atrophy. Color fundus photographs with the selected FPF analysis area highlighted in black (8A, 9A, 10A), corresponding FPF images (8B, 9B, 10B), and FPF images after anisotropic diffusion filtration (8C, 9C, 10C) of 71- (8A, 8B, 8C), 79- (9A, 9B, 9C), and 83- (10A, 10B, 10C) year-old eyes with advanced dry age-related macular degeneration and geographic atrophy.

To assess heterogeneity in FPF images, vertical line scan analysis and anisotropic diffusion filter analysis were used on all FPF images of the macula. Vertical line scan analysis resulted in 400 vertical slices across the rectangular FPF area of analysis. The mean FPF grayscale pixel intensity was calculated for each vertical slice (Fxn). The standard deviation of the mean FPF vertical slice intensities (Fx1, Fx2, Fx3, …, Fx400) was used to quantify heterogeneity in the analyzed macular area. All FPF images also underwent anisotropic diffusion analysis, which is a filtering technique that reduces background noise and brings out edges, lines, or other details in digital images.30

Because the grayscale intensities of each FPF image fall within a narrow range that often does not overlap with ranges of other FPF images, it is not possible to display all FPF images on the same relative fluorescence scale. Thus, to better visualize heterogeneity, the FPF images are shown at maximum contrast. Maximum contrast clips the extremes of white and black pixels by 0.5% in each image. Then, the remaining lightest and darkest pixels in the image are mapped and adjusted to pure white (level 255) and pure black (level 0). This makes the highlights appear lighter and shadows appear darker, but adjusts the maximum brightness and darkness to be the same in each image, and thus the fluorescence in the images is not plotted relative to each other but is plotted on the same scale. All data are expressed as the mean ± standard error of the mean, except for vertical line scan analysis, which is expressed as mean ± standard deviation because the number of vertical slices analyzed was the same for each sample. Differences were calculated using analysis of variance and t tests and a P value of less than .05 was considered statistically significant.

Results

Within the rectangular FPF analysis area (Figs. 2A, 3A, and 4A), the control patients showed little qualitative heterogeneity in the standard FPF images (Figs. 2B, 3B, and 4B) or in the images resulting from anisotropic diffusion filtration (Figs. 2C, 3C, and 4C). In the eyes with dry AMD (Figs. 5, 6, 7, 8, 9, and 10), localized hyperfluorescence was visible in one (Figs. 6B and 6C) of the three eyes without GA (Figs. 5, 6, and 7) and in all three eyes with GA (Figs. 8, 9, and 10). In the eyes with AMD with GA, visible FPF hyperfluorescence was present within the GA regions, at the atrophic margins, and beyond these margins, involving the surrounding retina (Figs. 8, 9, and 10). These regions of FPF hyperfluorescence were highly reproducible by the multiple FPF imaging of each eye.

Quantitatively, corrected mean average intensity of the rectangular FPF analysis area was significantly increased in eyes with AMD with (P = .044) and without (P = .00060) GA when compared to that of age-matched control eyes (Fig. 11). The standard deviations of FPF vertical slice intensities (Fx1, Fx2, Fx3, …, Fx400) for eyes with AMD were significantly greater compared to those of age-matched control eyes (Table 2) (P = .020). The standard deviations of FPF vertical slice intensities of the three eyes with AMD with GA, 19, 41, and 32 (Table 2), correlated well with the GA area in each of these eyes, 1.3, 7.5, and 2.3 mm2, respectively (Figs. 8A, 9A, and 10A).

Mean retinal flavoprotein fluorescence (average intensity ± standard error of mean) of three age-matched control, three moderate dry age-related macular degeneration, and three eyes with advanced dry age-related macular degeneration and geographic atrophy. Flavoprotein fluorescence levels are adjusted for reduced retinal mitochondrial mass in age-related macular degeneration.19 *P < .05, ***P < .001, compared with corresponding control.

Figure 11. Mean retinal flavoprotein fluorescence (average intensity ± standard error of mean) of three age-matched control, three moderate dry age-related macular degeneration, and three eyes with advanced dry age-related macular degeneration and geographic atrophy. Flavoprotein fluorescence levels are adjusted for reduced retinal mitochondrial mass in age-related macular degeneration.19 *P < .05, ***P < .001, compared with corresponding control.

Vertical Line Scan Analysis of Flavoprotein Fluorescence (FPF) Images (Standard Deviation of Fx1–400)a

Table 2: Vertical Line Scan Analysis of Flavoprotein Fluorescence (FPF) Images (Standard Deviation of Fx1–400)

One eye with AMD and GA (Fig. 8) showed visible FPF hyperfluorescence (Fig. 8B) yet had a normal, quantitative pattern when examined with vertical line scan analysis (Table 2); on the other hand, one eye with AMD without GA (Fig. 5) showed abnormal characteristics in vertical line scan analysis (Table 2) but no visible differences in FPF heterogeneity (Fig. 5B) from age-matched control FPF images (Figs. 2B, 3B, and 4B).

Discussion

The FPF hyperfluorescence inside the central, AMD-related GA regions has been an unexpected finding because these regions have a small number of RPE and photoreceptor cells capable of emitting FPF from their mitochondria. This observation may be explained by post-mortem studies of eyes with AMD. In fact, Dunaief et al.20 documented that eyes with GA showed increased apoptosis (TUNEL staining) in both the choroid and the outer and inner nuclear layers of the neurosensory retina, suggesting that metabolic dysfunction is widespread in the case of AMD-related GA and involves multiple chorioretinal structures. In eyes affected by GA, Kim et al.31 observed outer nuclear, inner nuclear, and ganglion cell layer loss of 77%, 9.5%, and 31%, respectively, in comparison with control eyes. Moreover, they distinguished that the outer, inner, and ganglion layer cell damage within the GA areas is even more pronounced in regions of significant RPE cell loss.31 These morphological changes support the contention that the FPF hyperfluorescence may be due to mitochondrial dysfunction of the overlying retina, and possibly the choroid, in atrophic AMD regions.

To further support that FPF is an indicator of mitochondrial stress, we conducted studies on human RPE cells exposed to sub-lethal oxidative stress and showed that increases in FPF inversely correlated with early reductions in mitochondrial membrane potential, an in vitro measurement that is indicative of mitochondrial dysfunction and increased mitochondrial membrane permeability.32 Furthermore, studies on primary cultures of human RPE cells and on fresh ex vivo human and rat neural retina exposed to oxidative stressors demonstrated that increases in FPF were reduced by antioxidants and abrogated by specific mitochondrial toxins, further indicating the mitochondrial origin of FPF.26,32,33 Although there are limitations in transferability of in vitro and ex vivo data to an in vivo context, the in vitro and ex vivo data strongly support that the FPF signal is indicative of mitochondrial dysfunction.

The human RPE cells and neural retinas used in the above studies did not contain significant lipofuscin. In eyes with AMD, variability in lipofuscin autofluorescence is present within GA lesions due to RPE cell dropout, release of lipofuscin granules, and lipofuscin accumulation in atrophic margins.34,35 The main autofluorescent component of accumulating lipofuscin in RPE cells is N-retinylidene-N-retinylethanolamine (A2E). A2E has been shown to inhibit the interaction between cytochrome c and cytochrome oxidase within the mitochondrial electron transport chain. Proper activation of cytochrome oxidase by cytochrome c is essential in producing hydrogen ions needed to drive ATP synthesis while chemically reducing molecular oxygen to water. Dysfunction of this process leads to increased oxidative stress that contributes to apoptosis.36 Accordingly, Vives-Bauza et al.37 showed that A2E causes reductions in RPE mitochondrial membrane potential, oxidative phosphorylation, and phagocytosis, all of which are abrogated by antioxidants. Thus, A2E, a key component of lipofuscin, is strongly implicated in mitochondrial dysfunction and oxidative stress at the sites of its accumulation, likely contributing to those elevated levels of oxidized flavoproteins detectable with FPF imaging.

Because we found FPF hyperfluorescence within the central GA areas, where lipofuscin hypofluorescence is characteristically found, it is unlikely that FPF in these areas is due to lipofuscin. However, it is possible that lipofuscin autofluorescence partly contributed to FPF hyperfluorescence at the margins of the atrophic lesions and in retinal areas with sub-RPE drusen deposits, where large amounts of lipofuscin are known to collect.34,35 Our method significantly reduces lipofuscin autofluorescence,24–27,33 but it uses wavelengths that overlap at the edges of the broad lipofuscin emission and excitation spectra. Thus, the lipofuscin signal is not entirely eliminated from the FPF signal.24–27,33 To more conclusively determine the contributions of lipofuscin to the FPF signal, a modified FPF instrument is being developed that includes a filter wheel with multiple filter sets. This will allow for both FPF and standard fundus autofluorescent imaging to be rapidly conducted on the same instrument, under optimal imaging conditions for each method, to obtain images with the same acquisition area and dimensions. Algorithms using ratio analyses will be developed to compare FPF and lipofuscin autofluorescence images to each other and correct for and eliminate the lipofuscin contributions in the FPF images. Additionally, to enable detection of more subtle heterogeneity and allow further discrimination between areas of retinal tissue with variations in mitochondrial dysfunction, more sophisticated algorithms for enhancement and quantitative analyses of FPF images are underway. Once the modified instrument and algorithms are developed, longitudinal studies will be conducted with patients with AMD with and without GA to compare retinal FPF and lipofuscin autofluorescent images before and after various AMD treatments.

Another interesting finding of this study was that one of the eyes with AMD without GA had localized FPF hyperfluorescence. It is possible that the localized hyperfluorescence could be indicative of mitochondrial dysfunction at the RPE and/or photoreceptor cell layers that may later develop into GA. Future longitudinal studies will be conducted to assess the utility of FPF as an early predictor of the development of disease complications, such as GA. Additionally, an unexpected finding was that two of the cases demonstrated conflicting qualitative and quantitative FPF data. One eye with AMD with GA had qualitatively visible FPF hyperfluorescence but a normal standard deviation in FPF vertical slice intensities, indicating that important information about disease status may be overlooked when quantitatively averaging information across the FPF image. Alternatively, one eye with AMD without GA had high standard deviation in FPF vertical slice intensities but no qualitatively visible differences in FPF heterogeneity, indicating that detection of subtle mitochondrial dysfunction in FPF images of eyes with AMD may require quantitative analysis. Therefore, our data demonstrate that qualitative and quantitative analyses of the FPF images may each provide important information about FPF heterogeneity that could possibly be missed when one method of analysis is used alone and, thus, both should be used when evaluating retinal FPF heterogeneity.

Because recent studies11–19 have implicated retinal mitochondrial dysfunction in the pathogenesis of AMD, an in vivo instrument to assess these metabolic alterations is necessary. In this study, even with a limited subset of patients, we have shown that FPF can be used to noninvasively detect AMD-induced retinal mitochondrial dysfunction, including heterogeneity within individual eyes. Because this method is not specific to AMD-induced mitochondrial dysfunction, it can be used to detect mitochondrial dysfunction caused by any mitochondrial stressor, as demonstrated in previous studies.24–27 Thus, retinal FPF cannot be a diagnostic measurement for AMD, but it can be used to qualitatively and quantitatively detect AMD-induced mitochondrial dysfunction. Future studies will be conducted to further assess the utility of FPF in detecting and evaluating AMD-induced metabolic alterations of the macula, including longitudinal studies on larger patient sets to determine the ability of FPF to monitor AMD disease progression and mitigation with treatment.

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Inclusion and Exclusion Criteria

Inclusion Criteria
• Adults between 65 and 85 years of age, capable of providing informed consent
• Confirmed diagnosis of unilateral or bilateral AMD either early stage or intermediate stage dry AMD (AREDS Category 2 or 3), or severe dry AMD (AREDS Category 4)
• For subjects with severe AMD, a confirmed diagnosis of unilateral or bilateral geographic atrophy compatible with AREDS Category 4 AMD, as well as the following criteria:
  – A well demarcated area of atrophy secondary to AMD
  – The total lesion being ⩽ 20 mm2 or 8 disc areas in diameter
  – If the lesion is multifocal, at least one of the focal lesions must be ⩾ 1.25 mm2 or 0.5 disc areas in diameter
Exclusion Criteria
• Exudative (“wet�?) AMD in either eye including any evidence of retinal pigment epithelial tears, retinal neovascularization, or vitreoretinal traction maculopathy
• Previous treatment with laser, photodynamic therapy, intravitreal injection, or previous treatment for AMD other than taking vitamins or other oral vitamin supplements
• Prior vitrectomy, penetrating keratoplasty trabeculectomy, or trabeculoplasty
• Lens removal in the 3 months prior to screening or any intraocular surgery within 6 months prior to imaging
• Other significant ophthalmologic disease (eg, glaucoma or diabetic retinopathy) or any condition that reduces the clarity of the media (eg, advanced cataract or corneal abnormalities)
• Any of the following medical conditions: diabetes mellitus, malignancy within the previous 5 years, documented HIV infection or other immunodeficiency syndrome, or other serious concomitant medical illness
• Systemic corticosteroids or immunosuppressive therapy within the 6-month period prior to imaging
• Unable to comply with requirements of the study protocol

Vertical Line Scan Analysis of Flavoprotein Fluorescence (FPF) Images (Standard Deviation of Fx1–400)a

Control EyesModerate AMD EyesAdvanced AMD Eyes with GA
Fig 2BFig 3BFig 4BFig 5BFig 6BFig 7BFig 8BFig 9BFig 10B
20.218.815.729.234.121.618.841.231.8

10.3928/15428877-20120712-02

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