Fundus Autofluorescence

Transcription

1 E-ISSN Major Review Fundus Autofluorescence Delhi J Ophthalmol 2013; 24 (2): DOI: * Vivek Pravin Dave, # Rajeev R. Pappuru * Consultant Retina, Uvea and Glaucoma, Netra Mandir Hi-Tech Eye Institute, Mumbai, India # Consultant and Head, Retina and Uvea Smt. Kanuri Santhamma Centre for Vitreo Retinal Diseases LV Prasad Eye Institute, Hyderabad, India *Address for correspondence Fundus Autofluorescence (FAF) is a unique in vivo method of fundus imaging. Although it has been an area of active research over the last half a century, its only in the past decade that major advances have been made in its clinical application. The imaging has its basis in the mapping of normal and abnormal fluorophores in various retinal conditions mainly lipofuschin. Recording of FAF is relatively easily accomplished, requires little time and is non-invasive. FAF signals are emitted across a broad wavelength spectrum. FAF imaging aids in identification and early detection of many retinal conditions often before changes can be picked up on other investigative modalities. FAF can assist in tracking temporal changes in fundus lipofuschin accumulation; detect pre clinical stage of fundus disorders related to lipofuschin, aid differential diagnoses based on different lipofuschin accumulation patterns and also in assessing risk factors that determine lipofuschin accumulation. FAF can also predict progression in various degenerative and age related retinal disorders before actual functional deficit occurs. In this review we shall discuss the principle and techniques of acquiring an FAF image. We shall describe a normal FAF picture with its basis and review various FAF features of various fundus pathologies. Many vitreo retinal diseases like age related macular degeneration, idiopathic macular telangiectasia, choroidal neovascular membranes, central serous chorioretinopathy among others have different autofluorescence patterns which will be discussed in this review. Keywords : fundus autofluorescence cslo fundus camera hypo autofluorescence hyper autofluorescence Vivek Pravin Dave MD, FRCS, DNB Consultant Retina, Uvea and Glaucoma Netra Mandir Hi-Tech Eye Institute, Mumbai, India vivekoperates@yahoo.co.in Introduction Lipofuschin is an intralysosomal polymer. It is primarily composed of cross linked protein units formed by ferrous-catalyzed oxidative processes. By its physical property, lipofuschin is not degradable. Also it cannot be excreted by the cells and hence accumulated in vivo. The amount of accumulation depends on the mitotic activity of the cells. Post mitotic cells accumulate more lipofuschin than rapidly dividing cells. The ferrous component of the lipofuschin moloecules sensitizes lysosomes to oxidative stress thus promoting apotosis. Lipofuschin also decreases local phagocytic activity and thus causing cell degeneration with increasing senescence. As lipofuschin is has, it had properties which aid in clinical examination of the fundus which has abundance of lipofuschin. Fundus autofluorescence has emerged as a new exciting tool for assessment of retinal, especially macular diseases and for their prognostication and follow up. The major source of fundus autofluorescence (FAF) in the eye is the accumulated lipofuschin in the retinal pigment epithelium (RPE). This FAF depends upon the renewal of the outer segment of the photoreceptors and the resultant balance between accumulation and clearance of lipofuschin. 1 Both RPE dysfunction/destruction and loss of photoreceptors cause decreased FAF as the source of lipofuschin is lost. 2 Principle and Technique Lipofuschin is composed of over 10 different flourophores and is not a single compact compound. RPE digests the tip of the photoreceptor outer segments by phagocytosis. A component of the digestive process which stays undigested is called lipofuschin. Though most of the components are not well characterized, A2E (N-retinyl- N-retinylidene ethanolamine), a bisretinoid compound is supposedly the best known fragment. 2 A2E has cellular toxicity and is immune to lysosomal degradation. Various noxious effects attributed to A2E are cellular membrane disruption, apotosis and DNA damage. It accumulates in senescent and diseased cells and gives rise to specific FAF patterns. The bisretinoids form in the RPE cells by random oxidative 80

2 Major Review Dave V P & Pappuru R R ISSN reactions involving isomers of vitamin A 11-cis-retinal and all trans retinal. FAF is obtained by stimulating the fundus with UV light or blue light in the wavelength range of 300nm to 600nm. The emission spectrum varies from 480nm to 800nm but is maximal in the 600nm to 640nm range. There are two different types of FAF known based on the absorption-emission spectrum of the fluorophores: the short wavelength autofluorescence (SW-FAF) and the near infrared autofluorescence (NIR-FAF). This classical type of FAF is called as short wavelength (SW-FAF) autofluorescence, which is based primarily on fluorescence from lipofuschin group of compounds. Recently, melanin which is a normal pigment found in the RPE cells and choroid is also found to have autofluorescence properties. Melanin has trophic properties and protects the RPE cells from oxidative and phototoxic damage. These melanin molecules are known to fluoresce by an excitation with 790nm wavelength but the image is times less intense than that of the SW-FAF. This is termed as the near-infrared (NIR-FAF) autofluorescence. As against lipofuschin in the SW-FAF, in the NIR-FAF, the AF originates from the melanin in the RPE. 3 The two commonly available modalities for capturing FAF are the cslo (confocal scanning laser ophthalmoscope) and the fundus camera. The cslo uses an excitation wavelength of 488nm and a barrier filter set at 500nm to block the reflected light and allow only the AF through. cslo continuously scans the retina with immediate digitization and display of the FAF image on the screen. As the modality uses light in the blue spectrum this can be discomforting to the patient. It is thus recommended to keep the cslo blue light exposure to the minimum. To achieve this, the initial focusing of the cslo camera should be in the near infrared reflectance mode. Once focused in the near infrared reflectance mode, the camera is then focused in the red free reflectance mode. Finally the barrier filter is set and the FAF image is obtained. A mean of 8-16 final images is taken and their pixel values normalized to give a final cslo based FAF image. In contrast to the laser scanner of cslo, the fundus camera uses a single flash and images the entire retinal area at the same time. It uses a band pass filter set at nm for excitation and nm as a barrier. Usage of this combination of wavelengths is important to negate the effect of the lens. Lipofuschin as such has the ability to fluoresce at a large range of wavelength from nm. At about nm, the crystalline lens has a range of peak fluorescence and hence interferes with an optimum AF image acquisition. This is because of the increase in blue-green fluorophores in the lens with age. With a band pass filter set at nm for excitation and nm as barrier, the effect of the lenticular AF is negated as the lens AF occurs at wavelengths shorter than the upper cut off of the band pass filter. The differences between the cslo and fundus camera system of AF image acquisition can be summarized as follows in (Table 1). Table 1. Comparison of cslo and fundus camera based AF cslo Fundus camera Excitation wavelength 488nm nm Barrier wavelength 500nm nm Wavelength absorption Absorbed by lens, blood Not absorbed by lens, blood Optic nerve Less fluorescence More fluorescence Back ground Contrast Better Less Nuclear sclerosis Less affected More affected Characterisitics of a normal FAF at the macula Normally, the optic nerve head appears typically dark as it is devoid of RPE and thus of lipofuschin (Figure 1). The retinal vessels also exhibit a near dark image due to absorption phenomenon by the blood. The fovea shows a distinct dark area of hypofaf with gradual increase in FAF peripheral to the fovea. 4 This is caused by absorption from luteal pigments (i.e., lutein and zeaxanthin) in the neurosensory retina and possible spatial differences in melanin deposition. Further away there is a uniform decremental FAF signal. Few examples of FAF in various retinal disorders are describes subsequently. Figure 1: Normal fundus autofluorescence Principles of abnormal AF An abnormal AF can either be artifactual or actual. Actual abnormal AF is due to a change in the composition of fluorophores or presence of autofluorescent material anterior to the RPE. Artifactual change in AF can be caused by media opacities. Lenticular opacities seem to the most interfering as the excitation barrier filter range can be affected by the degree of nuclear sclerosis. As explained above, the cslo imaging and the newer fundus cameras have different in built mechanisms to negate the effect of lenticular sclerosis. FAF signal can be reduced either due to decrease in RPE lipofuschin density, increased RPE melanin content or absorption of signals from extracellular material or fluid from in front of the RPE. RPE density decreases in RPE atrophy and in hereditary retinal dystrophies. RPE melanin is increased in conditions of RPE hypertrophy which masks 81 Del J Ophthalmol 2013;24(2)

3 E-ISSN Fundus Autofluorescence Major Review the signals emitted by the lipofuschin. Conditions like intra retinal edema, intraretinal and pre retinal hemorrhages and scars all reduce the FAF signal intensity. Media opacities in the cornea, lens or vitreous also reduce the FAF signals. FAF signals may be increased in conditions of excessive RPE lipofuschin accumulation, excessive fluorophores around the RPE layer or in cases of depletion of signal absorbing material. Excessive lipofuschin accumulation is known to occur in conditions like age related macular degeneration, Stargardts dystrophy, adult vitelliform macular dystrophy. Accumulation of fluorophores around the RPE occurs in macular edema, pigment epithelial detachments, old intraretinal and sub retinal hemorrhages and in choroidal nevi and melanomas. In this review we describe in detail a few common conditions faced in the clinic with respect to their FAF patterns. Central serous chorioretinopathy Central serous chorioretinopathy (CSCR) is characterized by leakage of fluid at the level of the RPE leading to serous retinal neuro sensory retinal detachment. The FAF in acute CSCR does not come from the lipofuschin as lipofuschin is an intra organelle material present within the RPE. These diseases are generally associated with physical separation of the photoreceptor outer segments from the RPE. The shed photoreceptor outer segments are theorized to accumulate on the outer retina and in the subretinal space. They contain retinoids and oxidized fatty acids which have ability to fluoresce. 5 In acute CSCR, the pattern could either be hyperaf or hypoaf depending on the time from onset. To begin with there are minimal autofluorescence changes in CSCR. with time there is slow increasing hyper AF noted. There is usually a small area of hypofaf at the site of the leakage 11,12 (Figure 2). This hypoaf is theorized to be due to the blockade of the fluorophore reflectance due to fluid or localized RPE destruction. The area of the neurosensory detachment is usually hypofluorescent to start with due to accumulated sub retinal fluid. As fluid accumulates, fluorophores aggregrate in it, increasing the AF with time. The FAF is usually uniformly distributed all over the pathology but in some cases it may be more pronounced inferiorly due to gravitation. OCT scanning corresponding to the FAF images shows reflective material aggregated on the outer surface of the retina. The amount of material aggregated is found to be corresponding to the strength of FAF. In chronic CSCR, areas of RPE atrophy develop which show hypo AF while areas of subretinal precipitates show hyper AF. 12 (Figure. 3) The borders of the descending tracts are also hyperaf. This hyperaf arises because the phagocytosis of the photoreceptor outer segments is compromised due to the lack of apposition with the RPE. Descending tracts of recent origin also show hyper AF. Sub retinal fibrin however shows no AF activity. Figure 2: Acute Central serous chorioretinopathy Figure 3: Chronic Central serous chorioretinopathy 82

4 Major Review Dave V P & Pappuru R R ISSN Dry AMD It has been demonstrated that drusens can exhibit increased, decreased, or normal autofluorescence intensities. Absence of a specific hypo or hyperaf pattern with the presence of drusens is attributed to the size of the drusens being < 60 microns or to the yellow macular pigment concentration. In case the drusens are crystallized, they have a hypoaf pattern due to absorption of the incident wavelengths. Usually drusens will show a mild hyperaf pattern due to the increased accumulation of lipofuschin. The FAF in the vicinity of drusens often varies. Areas of pigment clumping or besides crystallized drusens may show an increased FAF. This usually occurs due to the presence of melanolipofuschin granules in the pigment clumps. Studies have also shown that drusenoid pigment epithelial detachments and confluent drusens cause a pattern of patchy mild hyperaf. 13,14 This patchy hyperaf corelates with a high risk of development of choroidal neovascularization at a later date and can thus be a predictor of future CNVM. Reticular drusens can be well picked up on FAF. They appear as patchy areas of decreased FAF intensity surrounded by a lacy pattern of normal FAF. Reticular drusen are usually more widespread over the superior macula close to the superior arcade vessel. With advanced disease, areas of geographic atrophy develop which show a loss of photoreceptors and RPE. The corresponding area shows dense hypoaf. A functional correlate has been demonstrated between the areas of hyperaf and reduced photoreceptor function in dry AMD. 15 (Figure 4) Functional correlation over time has proven that new areas of geographic atrophy and spread of pre existing atrophy occurs only in areas that showed abnormally high levels of AF previously. 9 Based on the FAF pattern at the junctional zone of the atrophic and normal retina the geographic atrophy FAF pattern is classified into several types. Eyes showing areas with increased FAF adjacent to the atrophic patch and also elsewhere are termed as having a diffuse pattern. Eyes with increased FAF only at the geographic atrophy margin are further classified as focal, banded and patch according to the distribution of the FAF. Eyes with banded and diffuse pattern are known to show greater amount of atrophy over time as compared to those with the other forms. CNVM AF patterns in CNVM have been less characterized than those in geographic atrophy. In the initial stages, the area of CNVM showing leakage on fluorescein angiogram may show no change in AF indicating preserved viability of the RPE locally. Purely Classic CNVM lesions show a decreased signal at the site of the CNVM caused by the blockade of FAF by the subretinally located CNVM tissue. This is usually a homogenously reduced FAF. In contrast, in Occult CNVM lesions, show heterogenous FAF due to irregular CNVM tissue located subrpe causing foci of RPE destruction.16 (Figure 5) Studies have shown that the abnormal area on AF is larger than that seen on the Figure 4: Dry AMD Figure 5: CNVM 83 Del J Ophthalmol 2013;24(2)

5 E-ISSN Fundus Autofluorescence Major Review angiogram thus hypothesizing that AF may better delineate the edge of an advancing CNVM rather than angiogram. As against in geographic atrophy, RPE lipofuschin may not play a major role in CNVM lesions as there is hardly any increased FAF noted surrounding the CNVM lesion. In end stage CNVM disease, a decreased FAF signal is noted primarily due to the local destruction of the RPE and hypo AF signal from the scar tissue. CME The FAF in CME shows hyperautofluorescent cysts corresponding to the cysts on the FFA and fundus photograph. These intraretinal cysts cause lateral displacement of the macular pigments resulting in a focal decrease in their concentration. 17 (Figure 6) Macular Hole Macular holes show a highly increased FAF. In macular holes, there is an absent overlying neurosensory retina and consequently absent luteal pigments. This allows the excitation and emission rays to pass to and fro the RPE easily. There is a fine ring of hypoaf just surrounding the hole due to the upturned edges of the macular hole which constitutes of tissue containing luteal pigments and also because the increased thickness there impedes the passage of the excitation rays. 18 Following a successful macular hole surgery, the FAF returns to a normal pattern. (Figure 7) Figure 7: Macular hole Fundus Flavimaculatus Fundus flavimaculatus/stargardt s dystrophy shows a central oval area of hypoaf surrounded by small discrete spots of hyperaf and hypoaf. The peripheral flecks are seen as hyper auto fluorescent spots. Histopathologically, the flecks are known to constitute of enlarged RPE cells with abundant lipofuschin thus explaining their AF behavior. 19 Advanced cases show complete area of hypoaf over the central macula. 20 (Figure 8) Figure 6: CME Best s vitelliform dystrophy The vitelliform material in Best s vitelliform dystrophy shows hyperaf. Any area of reduced FAF signal noted is suggestive of incipient RPE atrophy. 21 (Figure 9) 84

6 Major Review Dave V P & Pappuru R R ISSN Retinitis Pigmentosa Retinitis pigmentosa shows a characteristic peripapillary area of hypoaf. The associated RPE changes in the peripheral fundus demonstrate varied areas of hypoaf and hyperaf. The macula shows a typical ring of increased FAF around the central area of hypoaf. 22 It has been shown that the size of the ring correlates with the functional field of vision. In some patients over time, this ring constricts and encroaches the fovea. 12 This is especially useful to distinguish cases with no clear cut clinical triad of RP (Figure 10) Figure 8: Fundus flavimaculatus Figure 10: Retinitis pigmentosa Idiopathic Macular Telangiectasia In idiopathic macular telangiectasia, there is a characteristic AF pattern of loss of the central normal hypoaf which is replaced by accentuated central hyperaf. 23 (Figure 11) In cases where there is deposition of pigment, the local area shows hypoaf or a complete lack of AF signal due to blockade of the signal by the dense pigment. Figure 9: Best Vitelliform dystrophy Choroiditis Choroiditis gives a varied AF pattern. The pattern depends upon the level of activity of the choroiditis. During active choroiditis, usually there is a hyperaf pattern. It is hypothesized that inflammation kindles certain oxidative pathways. Activation of these oxidative pathways o to an increase in fluorophore content in the RPE cells. 85 Del J Ophthalmol 2013;24(2)

7 E-ISSN Fundus Autofluorescence Major Review Figure 12: Active choroiditis This causes hypoaf signal in the area of healed choroiditis. (Figure 13) Secondary complications like inflammatory CNVM demonstrate AF patterns akin those seen in age related CNVM. Figure 11: Idiopathic macular telangiectasia Inflammation is also hypothesized to cause RPE hypertrophy and RPE hyperplasia. This in turn causes a hyper AF signal (Figure 12). Active inflammation also causes oxidation of the melanin in the RPE. This oxidized melanin leads to the formation of specific fluorophores whose absorptionemmision spectra resemble that of lipofuschin, thus increasing AF signal. 24,25 In healed choroiditis, the involved RPE cells show scarring and in the process they contract. This makes the surrounding area bereft of RPE cells and the consequent lipofuschin. 86

8 Major Review Dave V P & Pappuru R R ISSN In conclusion, FAF is a unique non invasive investigation which adds further information to the conventional angiography and OCT used for retinal disease evaluation. As the technique involves we may see FAF replacing other invasive investigations in certain disorders. Financial & competing interest disclosure The authors do not have any competing interests in any product/ procedure mentioned in this study. The authors do not have any financial interests in any product / procedure mentioned in this study References Figure 13: Healed choroiditis 1. Dorey CK, Wu G, Ebenstein D, et al. Cell loss in the aging retina. Relationship to lipofuscin accumulation and macular degeneration. Invest Ophthalmol Vis Sci 1989; 30: Feeney-Burns, L, Hilderbrand, ES, Eldridge, S. Aging human RPE: Morphometric analysis of macular, equatorial, and peripheral cells. Invest Ophthalmol Vis Sci 1984; 25: Bui TV, Han Y, Radu RA, Travis GH, Mata NL. Characterization of native retinal fluorophores involved in biosynthesis of A2E and lipofuscin-associated retinopathies. J Biol Chem 2006; 281: Sparrow, JR, Fishkin, N, Zhou, J et al. A2E, a byproduct of the visual cycle. Vision Res 2003; 43: Sparrow, JR, Nakanishi, K, Parish, CA The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 2000; 41: Sparrow, JR, Zhou, J, Ben-Shabat, S et al. Involvement of oxidative mechanisms in blue-light-induced damage to A2Eladen RPE. Invest Ophthalmol Vis Sci 2000; 43: Holz, FG, Bellmann, C, Margaritidis, M, et al. Patterns of increased in vivo fundus autofluorescence in the junctional zone of geographic atrophy of the retinal pigment epithelium associated with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 1999; 237: Sparrow, JR, Zhou, J, Cai, B. DNA is a target of the photodynamic effects elicited in A2E-laden RPE by blue-light illumination. Invest Ophthalmol Vis Sci 2003; 44: Keilhauer, CN, Delori, FC Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin. Invest Ophthalmol Vis Sci 2006; 47: Steffen Schmitz et al. Fundus autofluorescence imaging. Review and perspectives. Retina 2008: 28: Spaide RF, Klancnik JM Jr. Fundus autofluorescence and central serous chorioretinopathy. Ophthalmology 2005; 112: Spaide RF. Autofluorescence from the outer retina and sub retinal space. Retina 2008; 28: Scholl, HP, Bellmann, C, Dandekar, SS, et al. Photopic and scotopic fine matrix mapping of retinal areas of increased fundus autofluorescence in patients with age-related maculopathy. Invest Ophthalmol Vis Sci 2004; 5: Holz, FG, Bellman, C, Staudt, S, et al. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci 2001; 42: McBain, VA, Townend, J, Lois, N. Fundus autofluorescence in exudative age-related macular degeneration. Br J Ophthalmol 2007r; 91: Epub 2006 Sep Dandekar SS, Jenkins SA, Peto, et al. Autofluorescence imaging of choroidal neovascularization due to age related macular degeneration. Arch Ophthalmol 2005; 123: Bessho K, et al. Macular autofluorescence in eyes with cystoid macular edema detected with 488-nm excitation but not with 580-nm excitation. Graefes Arch Clin Exp 2009; 247: von Rückmann, A, Fitzke, FW, Gregor, ZJ Fundus autofluorescence in patients with macular holes imaged with a laser scanning ophthalmoscope. Br J Ophthalmol 1998; 82: Steinmetz, RL, Garner, A, Maguire, JI, Bird, AC Histopathology of incipient fundus flavimaculatus. Ophthalmology 1991; 98: Lois, N, Halfyard, AS, Bird, AC, et al. Fundus autofluorescence in Stargardt macular dystrophy-fundus flavimaculatus. Am J Ophthalmol 2004; 138: Jarc-Vidmar, M, Kraut, A, Hawlina, M. Fundus autofluorescence imaging in Best s vitelliform dystrophy. Klin Monatsbl Augenheilkd 2003; 220: Robson, AG, Egan, CA, Luong, VA, et al. Comparison of fundus autofluorescence with photopic and scotopic finematrix mapping in patients with retinitis pigmentosa and normal visual acuity. Invest Ophthalmol Vis Sci 2004; 45: Helb HM, et al. Abnormal macular pigment distribution in Type 2 idiopathic macular telangiectasia. Retina 2008; 28: Kayatz P, Thumann G, Luther TT, Jordan JF, Bartz-Schmidt KU, Esser PJ, Schraermeyer U. Oxidation causes melanin fluorescence. Invest Ophthalmol Vis Sci 2001; 42: Spaide RF. Autofluorescence imaging of acute posterior multifocal placoid pigment epitheliopathy. Retina 2006; 26: Del J Ophthalmol 2013;24(2)