Retinal flavoprotein autofluorescence as a measure of retinal health

Abstract

Purpose: To establish that increased autofluorescence of mitochondrial flavoproteins, an indicator of mitochondrial oxidative stress, correlates with retinal cell dysfunction.

Methods: Retinal flavoprotein autofluorescence (FA) was imaged in humans with a fundus camera modified with 467DF8-nm excitation and 535-nm emission filters and a back-illuminated, electron-multiplying, charge-coupled device camera interfaced with a computer equipped with customized image capture software. Multiple digital images, centered on the fovea, were obtained from each eye. Histograms of pixel intensities in grayscale units were analyzed for average intensity and average curve width. Adults with diabetes mellitus, age-related macular degeneration (ARMD), central serous retinopathy, and retinal dystrophies, as well as healthy control volunteers, were imaged. Monolayers of cultured human retinal pigment epithelial (HRPE) cells, HRPE cells exposed to sublethal doses of H2O2, and HRPE cells exposed to H2O2 in the presence of antioxidants were imaged for FA using fluorescent photomicroscopy.

Results: Control patients demonstrated low levels of retinal FA, which increased progressively with age. Diabetics without visible retinopathy demonstrated increased FA levels compared to control volunteers (P < .001). Diabetics with retinopathy demonstrated significantly higher FA values than those without retinopathy (P < .04). Patients with ARMD, central serous retinopathy, or retinal dystrophies also demonstrated significantly increased FA. Compared to control RPE cells, cells oxidatively stressed with H2O2 had significantly elevated FA (P < .05), which was prevented by antioxidants (P < .05).

Conclusions: Retinal FA is significantly increased with age and diseases known to be mediated by oxidative stress. Retinal FA imaging may provide a novel, noninvasive method of assessing retinal health and retinal dysfunction prior to retinal cell death.

Figures

FIGURE 1

Bar graph of average pixel intensities for retinal flavoprotein autofluorescence (FA) from control volunteers in 3 age groupings. Three volunteers were included in each age group, with all volunteers within 1 to 2 years of the group’s average age. Control volunteers in the 25-year-old group had significantly less retinal FA than either the 45-year-old group (P = .04 ) or the 60-year-old group (P = .01). gsu, grayscale units.

FIGURE 2

Histograms of retinal flavoprotein autofluorescence (FA) pixel intensities from 4 age-matched patients demonstrating increased pixel intensity in diabetes mellitus and particularly in diabetic retinopathy. Four histograms from each eye (right = blue, left = red) are shown. Far left panel, 59-year-old control volunteer without ocular disease or history of diabetes (control). Middle left panel, 61-year-old patient without ocular disease or known history of diabetes mellitus. Increased retinal FA, however, prompted serum glucose testing, which was found to be abnormal. Middle right panel, 63-year-old patient with diabetes mellitus without retinopathy. Far right panel, 61-year-old patient with bilateral nonproliferative diabetic retinopathy. Right shift of histograms denotes increased retinal FA intensity. Broader curve width denotes greater cellular variability in FA intensity within the 3° field that is imaged. AI, average pixel intensity, ACW, average curve width.

FIGURE 3

Bar graph of average pixel intensities from patients with diabetic retinopathy (aged 56 to 68, n = 7), diabetics without retinopathy (aged 54 to 67, n = 7), and age-matched nondiabetic controls (aged 57 to 67, n = 7). Patients with diabetes demonstrated significantly increased retinal flavoprotein autofluorescence (FA) pixel intensity compared to nondiabetic control volunteers (P = .001). Diabetics with retinopathy demonstrated yet higher intensities of retinal FA compared to diabetics without retinopathy (P = .04). gsu, grayscale units.

FIGURE 4

Retinal flavoprotein autofluorescence (FA) pixel intensity histograms from 77-year-old patient with age-related macular degeneration (ARMD) (left) and 67-year-old control patient without visible clinical findings of ARMD (right). ARMD patient had history of resolved exudative ARMD with VA 20/50 (best corrected) OD and dry ARMD with geographic atrophy and VA 20/400 (best corrected) OS. Four histograms of the right (blue) and left (red) eye from each patient are presented. Right shift of histograms denotes increased retinal FA intensity. Broader curve width denotes greater cellular variability in FA intensity within the 3° field that is imaged. AI, average pixel intensity; ACW, average curve width; gsu, grayscale units.

FIGURE 5

Flavoprotein autofluorescence (FA) pixel intensity histograms from 49-year-old patient with bilateral central serous retinopathy (left) and age-matched control (right). Four histograms of the right (blue) and left (red) eye from each patient are presented. Right shift of histograms denotes increased retinal FA intensity. AI, average pixel intensity; ACW, average curve width; gsu, grayscale units.

FIGURE 6

Flavoprotein autofluorescence (FA) pixel intensity histograms from 36-year-old patient with retinitis pigmentosa (left) and age-matched control (right). Four histograms of the right (blue) and left (red) eye from each patient are presented. Right shift of histograms denotes increased retinal FA intensity. AI, average pixel intensity; ACW, average curve width.

FIGURE 7

Flavoprotein autofluorescence (FA) production induced by hydrogen peroxide (H2O2) (left) and C2-ceramide (right) in human retinal pigment epithelial (RPE) cells. Left, Effect of ROS scavenger on H2O2-induced FA production. RPE cells were preincubated with or without N-acetylcysteine (NAC; 1 mM) for 30 minutes and then treated with H2O2 (0.2 mM) in the presence and absence of NAC for 3 hours. Right, Effect of inactive form of C2-ceramide, dihydroceramide C2 on C2-ceramide-induced FA production. RPE cells were preincubated with or without dihydroceramide C2 (50 μM) for 30 minutes and then treated with C2-ceramide (50 μM) in the presence and absence of dihydroceramide C2 for 2 hours. Flavoprotein autofluorescence was quantitated with a FlexStation fluorescence plate reader with excitation and emission wavelengths set to 450 nm and 520 nm, respectively. Data are presented as mean ± SEM. N = 3 cell lines. **P < .001, compared with control; ##P < .01, compared with H2O2-stimulated cells. *P < .05, compared with control; #P < .05, compared with C2-ceramide-stimulated cells.