Fluorescence Lifetime Imaging in Retinal Artery Occlusion

Abstract

Purpose.: Fluorescence lifetime imaging ophthalmoscopy is a technique to measure decay times of endogenous retinal fluorophores. The purpose of this study was to investigate fluorescence lifetimes in eyes with central and branch retinal artery occlusion.

Methods.: Twenty-four patients with central or branch retinal artery occlusion were included in this study. The contralateral unaffected fellow eye was used as control. Measurements were performed using a fluorescence lifetime imaging ophthalmoscope based on a HRA Spectralis system. Fluorescence excitation wavelength was 473 nm, and mean lifetimes were measured in a short (498–560 nm) and in a long (560–720 nm) spectral channel. Fluorescence lifetimes in the area of retinal artery occlusion were measured and compared to corresponding areas in contralateral unaffected eyes. Additionally, findings were correlated to optical coherence tomography measurements.

Results.: Retinal lifetime images of 24 patients with retinal artery occlusion were analyzed. Mean retinal fluorescence lifetimes were prolonged by 50% in the short and 20% in the long spectral channel in ischemic retinal areas up to 3 days after retinal artery occlusion compared to the contralateral unaffected eyes. In the postacute disease stage there was no difference between the lifetimes of affected areas and unaffected fellow eyes.

Conclusions.: Retinal artery occlusion leads to significantly longer fluorescence lifetimes of the retina in the acute phase and may serve as a useful indicator for acute ischemic retinal damage.

Retinal artery occlusion (RAO) is a common cause of severe visual loss.^1–3 It is classified into central retinal artery occlusion (CRAO) due to occlusion of the central retinal artery or into branch retinal artery occlusion (BRAO) when only a branch of the central retinal artery is affected.³ Painless sudden loss of vision, in conjunction with ophthalmoscopically visible retinal opacification in the acute phase, is the most important clinical sign of RAO.⁴ Optical coherence tomography (OCT) shows swelling of the inner retinal layers, predominantly the inner plexiform and inner nuclear layer, corresponding to the region supplied by the retinal arteries.^5,6 When the swelling has resolved after 2 to 3 weeks, the retinal transparency is restored as well. The postacute phase is characterized by varying degrees of inner retinal atrophy, vascular thinning, and optic nerve head pallor.⁷ On a more molecular level, ischemia causes retinal ganglion cells to switch to an anaerobic metabolism resulting in production of lactic acid.⁸ As a consequence, there is an increase in intracellular calcium levels, and extracellular concentrations of glutamate and aspartate increase markedly.⁹ Furthermore, a recent study has shown that high levels of Nox1 NAD(P)H (oxidized and reduced nicotinamide adenine dinucleotide) oxidase subunits in retinal ganglion cells generate reactive oxygen species (ROS).¹⁰ Whereas conventional techniques such as OCT or fluorescein angiography accurately image structural changes in RAO, an imaging technique that would allow viewing of metabolic changes may provide insights into the pathophysiology of retinal ischemia. 

In the last decade, fluorescence lifetime imaging microscopy (FLIM) has emerged as an important tool to study intracellular ion concentrations such as Ca²⁺ in vivo,¹¹ NAD(P)H photochemistry inside living tissue,^12,13 and FAD (oxidized flavin adenine dinucleotide)¹⁴ or to detect molecular interactions by the use of energy transfer.¹⁵ 

Fluorescence lifetimes represent the time a molecule spends in the excited state after excitation with laser light before returning to the ground state. These lifetimes are characteristic for each molecule and insensitive to its concentration, but responsive to the local tissue microenvironment.^16,17 The FLIM technique has already been used to identify transient focal ischemia in a model of cerebral ischemia. In this model, a 670-nm laser was used for excitation of endogenous fluorophores, which displayed a significantly prolonged lifetime in the ischemic hemisphere when compared to the uninjured hemisphere at 700-nm emission wavelength.¹⁸ 

Similar to FLIM, fluorescence lifetime measurement in ophthalmology (FLIO) has recently emerged as a technique to measure fluorescence lifetimes in the retina. Analogously to FLIM, decay times of natural fluorophores of the retinal tissue are measured after excitation with a laser.¹⁹ The FLIO technique has been shown to be highly reproducible, noninvasive, and easily deployable in a clinical setting.²⁰ 

The purpose of this study was to investigate whether ischemic areas after RAO display altered fluorescence lifetimes when compared to the unaffected contralateral eye. 

Methods

This prospective study was conducted with the approval of the local ethics committee and in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants before study entry. This study is registered at ClinicalTrials.gov as Measurement of Retinal Auto Fluorescence with a Fluorescence Lifetime Imaging Ophthalmoscope (FLIO Group) with the identifier number NCT01981148. 

Patients

Twenty-four patients after RAO were recruited between February 2013 and October 2014 at the Department of Ophthalmology at the University Hospital of Bern. Patients with other retinal disease and/or significant lens opacities were excluded from the study. 

Patients were assigned to either an acute group when artery occlusion was less than 3 days old, or to a postacute disease stage group when artery occlusion was more than 30 days old. 

All patients underwent a general ophthalmologic examination, and best-corrected visual acuity (Early Treatment Diabetic Retinopathy Study [ETDRS] letters) was measured. Fluorescence lifetime images were obtained after maximal pupil dilation with tropicamide 0.5% and phenylephrine HCl 2.5%. The two eyes were measured consecutively. The unaffected fellow eye was used as control eye. 

From every patient, fundus color images (Zeiss FF 450plus; Zeiss, Oberkochen, Germany) and OCT scans of the macula (Heidelberg Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany) were obtained as part of the general examination and disease documentation. 

Fluorescence Lifetime Imaging Ophthalmoscope

Retinal fluorescence lifetime imaging was performed using a fluorescence lifetime imaging ophthalmoscope based on a HRA Spectralis system (Heidelberg Engineering). Basic principles of the FLIO technique have been described previously.²⁰ Here we provide only a brief summary of this technique. 

For excitation of retinal autofluorescence, a 473-nm pulsed laser was used, raster scanning the central fundus with a repetition rate of 80 MHz. The emitted fluorescence photons were detected by highly sensitive hybrid photon-counting detectors (HPM-100-40; Becker & Hickl, Berlin, Germany) in a short (498–560 nm) and in a long (560–720 nm) spectral channel and registered by time-correlated single-photon counting (TCSPC) modules (SPC-150; Becker & Hickl). Eye tracking using the infrared reflectance image allows for data accumulation over the exposure duration of approximately 90 seconds that is required for the FLIO measurement. 

Recorded lifetime values were approximated by Becker & Hickl software (SPCImage 4.6) using a biexponential decay model and a binning factor of 1, as described previously.²⁰ 

For both spectral channels, for each recorded pixel the mean fluorescence lifetime Tm was calculated from the short and long lifetime components T1 and T2 and their respective amplitudes α1 and α2. 

Statistical Analysis

Mean fluorescence lifetime values were averaged within the areas of a standard ETDRS grid using the FLIO reader (ARTORG Center for Biomedical Engineering Research, University of Bern, Switzerland). Early Treatment Diabetic Retinopathy Study circle diameters were 1 mm for the central area, 3 mm for the inner ring, and 6 mm for the outer ring. 

Prism GraphPad commercial software package (Prism 6; GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. For CRAO, mean lifetime values from the ETDRS center, inner ring, and outer ring were compared to those from the contralateral unaffected fellow eye by Wilcoxon matched-pairs signed rank test (two-tailed, confidence interval 95%). For BRAO, affected areas were defined in the OCT and infrared images, and corresponding ETDRS grid areas were compared to the respective areas in the unaffected fellow eye. Analysis was done for both spectral channels. P values less than 0.05 were considered to be statistically significant. 

Results

A total of 24 patients with artery occlusion were enrolled in this study. Thirteen patients were tested after CRAO, with 11 patients in the acute stage and four patients in the postacute disease stage. Two patients were examined twice in both the acute and the postacute disease stages. Eleven patients were analyzed after BRAO, with seven patients in the acute stage and four patients in the postacute disease stage (demographic and clinical data are shown in the Table). 

Table

 

Demographics, Visual Acuity, and Disease Duration

 

Fundus Autofluorescence Lifetimes After CRAO

In the acute disease stage 1.6 ± 0.3 days after CRAO (mean ± standard error of the mean [SEM]), patients with CRAO displayed a characteristic fundus appearance with whitening of the retina and thickening of the inner retinal layers in OCT due to ischemia, and relative autofluorescence intensity was reduced in the ischemic areas by 25% in the short spectral channel (SSC, P = 0.001) and 38% in the long spectral channel (LSC, P = 0.002) (Supplementary Fig. S1A). In the control eyes, the LSC (560–720 nm) detected 84% more photons during the acquisition time compared to the SSC. In the CRAO eyes, this difference was 52%. Corresponding mean fluorescence lifetime values were prolonged in all ETDRS areas in both spectral channels compared to the corresponding area in the unaffected fellow eyes (Fig. 1; for further examples see Supplementary Fig. S2A). This difference was statistically significant in the inner and the outer ring of the ETDRS grid (P = 0.001) (Fig. 2A). In the inner ETDRS ring, mean lifetimes after CRAO were prolonged by 59% (209 ps) and 22% (89 ps) in the SSC and the LSC, respectively. In the central area of the ETDRS grid, mean lifetimes were nonsignificantly prolonged by 127 ps (SSC, P = 0.067) and 55 ps (LSC, P = 0.32). 

Figure 1

 

Ocular fundus of a 59-year-old patient 1 day after central retinal artery occlusion (left eye, CRAO) and the unaffected fellow eye. Fundus color image, autofluorescence image, fluorescence lifetime images in the short (SSC, 498–560 nm) and in the long (LSC, 560–720 nm) spectral channel (color scale: 200–600 ps). Arrows highlight retinal areas with prolonged fluorescence lifetimes.

Figure 2

 

Quantitative analysis of fluorescence lifetimes of the ocular fundus after central retinal artery occlusion (CRAO). (A) Acute (1.6 ± 0.3 days) and (B) postacute (3.4 ± 1.1 months) disease stage after CRAO. Mean fluorescence lifetime values are averaged within the ETDRS grid areas center (C), inner ring (IR), and outer ring (OR) and compared to the corresponding area of the unaffected fellow eye. Data are shown for the short (498–560 nm) and the long (560–720 nm) spectral channel (median ± interquartile range [IQR]); ***P value < 0.001, ns, not significant; n = 11 (acute) and n = 4 (postacute).

In four patients, fluorescence lifetime measurement was performed in a postacute disease stage 3.4 ± 1.1 months after CRAO (Supplementary Fig. S2B). Measured mean fluorescence lifetime values at this disease stage were comparable to the corresponding ETDRS areas in the control eyes in both spectral channels (P = 1 and 0.88) (Fig. 2B). 

Fundus Autofluorescence Lifetimes After BRAO

Patients with BRAO showed whitening consistent with retinal edema in the affected areas. In the OCT, a swelling of the inner retinal layers was measured in the acute stage (2.4 ± 0.9 days after BRAO) followed by an atrophy of affected retinal layers in the postacute disease stage 20.4 ± 17.6 months after BRAO (Figs. 3, 4B). In the acute phase, fluorescence lifetimes were prolonged by 50% (162 ps) in areas affected with BRAO in the SSC (P = 0.001) (Figs. 4A, 4C; Supplementary Fig. S3A). In the postacute disease stage, there was no difference between areas after BRAO and unaffected control areas (P = 0.94) (Figs. 4A, 4C; Supplementary Fig. S3B). 

Figure 3

 

Ocular fundus after inferior branch retinal artery occlusion (BRAO). Acute (1 day, upper row) and postacute (3 months, lower row) disease stage after inferior BRAO. Autofluorescence image with indicated line of the vertical optical coherence tomography (OCT) scan beside and fluorescence lifetime images of the short (SSC, 498–560 nm) and the long (LSC, 560–720 nm) spectral channel (color scale: 200–600 ps).

Figure 4

 

Quantitative analysis of fluorescence lifetimes of the ocular fundus and total retinal thickness after branch retinal artery occlusion (BRAO). (A) Mean fluorescence lifetime values of the short spectral channel and (B) mean retinal thickness (μm) of affected ETDRS grid areas (inner ring) in the acute (2.4 ± 0.9 days) and postacute (20.4 ± 17.6 months) disease stage are compared to corresponding areas in the unaffected fellow eye (median ± IQR); ***P value < 0.001, **P value < 0.01, *P value < 0.05, ns, not significant; n = 7 (acute) and 4 (postacute). (C, D) Representative fluorescence lifetime images of an acute (C) and postacute (D) disease stage after inferior BRAO in the right eye with unaffected fellow eyes beside. The inferior area of the inner ring of the ETDRS grid, which was used for quantitative analysis of fluorescence lifetime data, is marked in boldface (C, center; IR, inner ring; OR, outer ring).

Fluorescence Lifetimes and Retinal Thickness After RAO

In order to assess whether the degree of the swelling correlated with the prolonged lifetimes, we measured the retinal thickness using OCT. Swelling of the inner retinal layers was observed in OCT in all ischemic areas, resulting in an increase of the total retinal thickness of the inner ring of the ETDRS grid from 322 ± 7 to 455 ± 34 μm in patients after CRAO and from 333 ± 5 to 396 ± 20 μm in ischemic areas after BRAO (P = 0.0004 and 0.0075, respectively; Supplementary Figs. S1A, S1B; Fig. 4B). At the postacute disease stage, the retinal thickness had decreased to 261 ± 34 μm in CRAO and 284 ± 12 μm in BRAO (P = 0.08 and 0.01, respectively). 

We did not find any significant correlation between the total retinal thickness and the mean fluorescence lifetime (SSC P = 0.41 and LSC P = 0.08 in the acute disease stage; Supplementary Figs. S1C, S1D). However, as there are probably multiple factors influencing the mean fluorescence lifetime, correlation with the retinal thickness as a single parameter might be insufficient. 

Lens Status

Of the 13 patients in the CRAO group, seven patients were pseudophakic and six patients were phakic. In the BRAO group, only phakic patients were included in this study. 

Even though phakic patients tended to have longer mean fluorescence lifetimes, especially in the SSC, no significant difference was found between the fluorescence lifetime measurements of pseudophakic and phakic patients (SSC P = 0.17 and LSC P = 0.35). 

Analysis of Individual Fluorescence Lifetime Components

Qualitative analysis of the short (T1) and the long (T2) decay component of the fluorescence lifetime decay curve displayed characteristic distribution histograms in both spectral channels (Fig. 5A). The fluorescence lifetime cloud in retinae after artery occlusion was shifted toward longer lifetimes. The altered retinal areas showed a clearly distinguishable decay cloud with longer fluorescence lifetime components of T2. Other anatomical locations, for example, the central fovea, the optic nerve head, and the vessels, showed specific T1 to T2 lifetime constellations (Fig. 5B). 

Figure 5

 

Distribution histograms of lifetime components after central retinal artery occlusion (CRAO, left side) and branch retinal artery occlusion (BRAO, right side). (A) Representative combined fluorescence lifetime and intensity image of a healthy retina and a retina after acute retinal artery occlusion (short spectral channel). Corresponding distribution histograms are shown beside for the short (T1) against the long (T2) lifetime components. (B) Selected retinal areas are represented in the distribution histogram as specifically discernible clouds. (B1) Macula, (B2) unaffected retina, (B3) ischemic retina after retinal artery occlusion, (B4) shape of the optic nerve head.

Discussion

We investigated retinal autofluorescence lifetimes in 24 patients after central or branch RAO. During the acute phase of RAO, FLIO measurement showed significantly longer lifetime values in ischemic retinal areas with a sparing of the fovea when compared with unaffected fellow eyes. In the SSC (498–560 nm), fluorescence lifetimes were prolonged by 59% after CRAO and 50% after BRAO and in the LSC (560–720 nm) by 22% and 21%, respectively. In the postacute disease stage after artery occlusion, there were no differences in fluorescence lifetimes between the diseased areas and the unaffected fellow eyes in either channel despite marked atrophy of the inner retinal layers in OCT. 

We and others have previously characterized fluorescence lifetimes in the healthy retina.^20,21 In normal conditions, the shortest lifetimes (260 ± 30 ps, mean ± SEM) derive from the fovea, probably due to the influence of macular pigment. Outside of the fovea, the fundus exhibits lifetimes in the range of 360 ± 25 ps, whereas the retinal vessels and the optic disc feature longer lifetimes (>450 ps). 

However, in order to evaluate FLIO as a technique to detect metabolic changes, it is necessary to analyze known retinal pathologies such as ischemia occurring after RAO. 

During cerebral ischemia in a mouse model, lifetimes were shown to increase in vivo.¹⁸ However, the reason for prolonged lifetimes in this model is not yet understood and has been attributed to breakdown of the blood–brain barrier and extravasation of fluorescent molecules.¹⁸ 

There are several factors that may explain our findings of prolonged lifetimes after RAO. In the last decades the pathophysiology of RAO has been studied extensively.^7,10,22–26 

On the cellular level, retinal ischemia will lead to several changes; after interruption of retinal blood supply, swelling of the mitochondria of ganglion cells and bipolar cells is the first observed change due to altered extra- and intracellular ionic balances.²⁷ The ganglion cells are especially sensitive to oxygen damage and show extensive swelling in OCT during acute ischemia.²³ 

On a molecular level, ischemia leads to inhibition of electron transport, decreased adenosine triphosphate (ATP) production, increased cell Ca²⁺, decrease in pH, and release of glutamate.^22,25 Cell death will also trigger gene activation leading to cytokine synthesis and accumulation of inflammatory cells.²⁵ Furthermore, ROS are known to accumulate after hypoxic cell death,^25,28 leading to oxidation of nucleic acids, proteins, and lipids, resulting in further neuronal apoptosis.²² 

Undersupply of oxygen after acute RAO leads to alterations in the cellular metabolism and potentially to changes in the redox pairs NAD+/NADH (oxidized and reduced nicotinamide adenine dinucleotide) and FAD/FADH2 (oxidized and reduced flavin adenine dinucleotide) for the oxidative phosphorylation of adenosine diphosphate (ADP) to ATP.²⁹ Whereas NAD+ and FADH2 are nonfluorescent, the reduced NADH and the oxidized FAD show specific fluorescence properties depending on the excitation wavelength and their protein-binding state.^30,31 However, as NADH has a peak excitation wavelength of 340 ± 30  nm, which is much lower than the 473-nm laser used for excitation in the FLIO, NADH lifetimes are unlikely to contribute to our measurements.²⁹ In addition, the fluorescence emission of NADH (458 nm) is below the detection limits of the short channel of the FLIO device.¹⁷ On the other hand, FAD, predominantly located within the mitochondria, shows an excitation wavelength of 450 nm and emits approximately 528 nm¹⁷ and therefore is well within the detection limits of the FLIO system. In tissue with oxygen undersupply such as after RAO, the redox pair FAD/FADH2 will be shifted toward the reduced FADH2. However, as FADH2 is relatively nonfluorescent, the shift toward FADH2 would lead to a decrease in the contribution of FAD. Oxidized flavin adenine dinucleotide displays a heterogeneous fluorescence intensity decay spectrum with a 7-ps contribution that is characteristic of ultrafast fluorescence quenching and a very long average lifetime of 3.13 ns (unbound) or 2.75 ns (bound).³² Therefore the reduction of the ultrafast lifetime contribution would lead to an increase of lifetimes after RAO,^17,29 whereas, conversely, a reduction in the long component would lead to a decrease in lifetimes. Due to the heterogeneous fluorescence intensity decay characteristics of FAD and the possible influence of fluorescence quenching, we can only speculate about the contribution of this endogenous chromophore; and further studies are necessary to dissect the influence of FAD on retinal fluorescence lifetime changes. 

Another possible explanation for the prolongation of the fluorescence lifetimes in the acute phase of RAO may be an autofluorescence masking or blockage effect by the swelling of the inner retinal layers. It has been shown in a previous report that RAO leads to decreased autofluorescence intensity.³³ As fluorescence lifetimes are independent of the fluorophore's concentration,^17,29 autofluorescence blockage may cause a relative attenuation of the short lifetimes of the retinal pigment epithelium and the photoreceptor layer and an increased contribution of longer autofluorescence lifetimes of the layers with retinal swelling due to ischemia. Furthermore, increased contribution of lens fluorescence due to increased scattering within the thickened retinal layers may lead to longer lifetimes, as the crystalline structure of the lens features fluorescence lifetimes of approximately 1300 ps.²⁹ This effect is most pronounced in the SSC (498–560 nm) where the influence of the lens is the highest. Therefore the mean measured lifetimes in areas with retinal swelling would appear longer than in the surrounding unaffected retinal tissue including the central retinal fovea. However, as we have observed changes in fluorescence lifetimes in pseudophakic patients similar to those in phakic patients, this may only marginally contribute to fluorescence lifetime changes in patients with RAO. 

Another interesting finding is that retinal fluorescence lifetimes did not show any differences compared to the contralateral unaffected eyes in the postacute disease stage after artery occlusion. This was observed despite the fact that total retinal thickness was significantly reduced at the affected retinal areas with diffuse atrophy of the inner retinal layers.⁵ These findings support the hypothesis that the short autofluorescence lifetimes, much like the autofluorescence intensity, are largely derived from the intact retinal pigment epithelium with lipofuscin as the main fluorophor and the photoreceptor layer.^34–37 

As metabolic changes go hand in hand with the swelling of the inner retinal layers, it is difficult to identify the specific contribution of each with regard to the prolonged lifetimes observed in our patients. 

This study has some limitations. Firstly, the number of patients within each subgroup, especially for the postacute disease stage, is small, and further studies with more patients need to be performed to confirm our findings. Additionally, longitudinal examinations within the same patients may provide additional information on the natural time course of fluorescence lifetimes. Furthermore, there is limited information available about the source, interaction, and contribution of retinal fluorescence lifetimes within the eye. As fluorescence decay times are highly dependent on their microenvironment, mean retinal lifetime might not be entirely explained by individual metabolic components. Previous reports have shown that fluorescence lifetime quenching may be used to measure oxygen tension in vivo in mice given 100% oxygen³⁸ and as such, hypoxia may contribute to our findings. Animal models or in vitro experiments using retinal pigment epithelium cells will be helpful to dissect the contribution of individual lifetime components to our findings in human RAO, and this is currently being investigated in our laboratory. 

In conclusion, significantly longer lifetimes were observed in areas affected with acute RAO and retinal swelling. Identified changes may be caused by altered cellular metabolism after retinal ischemia. 

Fluorescence lifetime measurements of the retina using a fluorescence lifetime imaging ophthalmoscope might be applied for diagnostic purposes and disease and therapy monitoring and may provide new clues for a better understanding of ischemic retinal diseases. 

Acknowledgments

Presented in part at the imaging conference of the Association for Research in Vision and Ophthalmology, Orlando, Florida, United States, May 3, 2014. 

Supported by a grant from the Swiss National Science Foundation (SNSF) (#320030_156019). The sponsor or funding organization had no role in the design or conduct of this research. 

Disclosure: C. Dysli, Heidelberg Engineering (F); S. Wolf, Heidelberg Engineering (F); M.S. Zinkernagel, Heidelberg Engineering (F)