Multimodal Imaging Including Spectral Domain OCT and Confocal Near Infrared Reflectance for Characterization of Outer Retinal Pa

Multimodal Imaging Including Spectral Domain OCT and Confocal Near Infrared Reﬂectance for Characterization of Outer Retinal Pathology in Pseudoxanthoma Elasticum

Peter Charbel Issa, Robert P. Finger, Frank G. Holz, and Hendrik P. N. Scholl

PURPOSE. To investigate the value of multimodal confocal scan- seudoxanthoma elasticum (PXE) is a rare systemic disease ning laser ophthalmoscopy (cSLO) for phenotyping fundus Pmainly affecting the cardiovascular system, skin, and eyes lesions in patients with pseudoxanthoma elasticum (PXE) and with a variable phenotype.1,2 The disorder with a prevalence to correlate these findings with morphologic alterations de- estimated to be 1 in 25,000 to 100,000 is a consequence of 3 tected by spectral domain optical coherence tomography (SD- mutations in the ABCC6 gene. Progressive fragmentation and OCT). calcification of elastic fibers in connective tissue result in pathologic changes most pronounced in the dermis, Bruch’s METHODS. Imaging was performed with a combined SD-OCT- membrane, and blood vessels.1,2 Characteristic lesions at the cSLO system (Spectralis HRA-OCT; Heidelberg Engineering, fundus are angioid streaks, peau d’orange, secondary choroidal Heidelberg, Germany). OCT scans were placed at locations of neovascularization, diffuse chorioretinal atrophy, and cho- interest on near-infrared (NIR) reflectance, fundus autofluores- rioretinal atrophic spots in the midperiphery, often with comet cence (FAF), and fluorescein angiography (FA) images. The tails pointing toward the posterior pole.1,2 Fundus lesions instrument allowed for exact topographic correlation of find- similar to those observed in pattern dystrophies have also been ings on OCT and cSLO images. described.4 So far, phenotypic investigation has been based RESULTS. NIR reflectance imaging showed the highest sensitiv- mainly on standard imaging methods such as ophthalmoscopy 2 ity to detect angioid streaks and peau d’orange compared to and fundus photography. However, fundus autofluorescence (FAF) imaging was recently found to reveal characteristic FAF or FA. On OCT scans, angioid streaks reliably showed 5,6 breaks in Bruch’s membrane. Peau d’orange was associated changes in patients with PXE. Other topographic fundus with alternating reflectivity within Bruch’s membrane. Charac- imaging modalities such as near-infrared (NIR) reflectance have never been used, and the relative value of specific imaging teristic mid-peripheral chorioretinal atrophies showed hypore- modalities for phenotyping retinal lesions in PXE remains un- flective spaces involving the outer neurosensory retina. In eyes known. It also remains to be determined whether such multi- with pattern dystrophy like alterations, subneurosensory accu- modal fundus imaging allows sensitive detection and charac- mulation of material was observed within areas of increased terization of fundus lesions in PXE. FAF. Histologic studies of fundus changes due to PXE are rare, CONCLUSIONS. SD-OCT in combination with cSLO imaging using and the underlying histologic alterations of such fundus lesions NIR light locates the primary pathologic formations of angioid remain largely unknown.7–12 High-resolution spectral domain streaks and peau d’orange in Bruch’s membrane. NIR reflec- optical coherence tomography (SD-OCT) has recently become tance imaging may be superior for detecting PXE-related fun- available and offers a quasi histologic assessment of the poste- 13–15 dus lesions at the level of Bruch’s membrane, because the blue rior ocular fundus in vivo. Systems combining SD-OCT laser light that is used in FAF and FA is markedly absorbed by with a confocal scanning laser ophthalmoscope (cSLO) allow the pigment epithelium and therefore may only detect alter- simultaneous recordings of cross-sectional OCT images with ations if this cell layer is also affected. The findings indicate that various topographic imaging modes such as NIR reflectance, multimodal cSLO and SD-OCT imaging of the outer retina FAF, and fluorescein angiography (FA) of the cSLO. Exact alignment of the SD-OCT scans with the topographic cSLO allows for screening of PXE related retinal alterations. (Invest images permits to correlate quasi in vivo histology with patho- Ophthalmol Vis Sci. 2009;50:5913–5918) DOI:10.1167/iovs.09- logic features observed on FAF, reflectance, and angiography 3541 images. The purpose of this study was to investigate PXE-related fundus lesions using multimodal cSLO imaging and to study From the Department of Ophthalmology, University of Bonn, whether SD-OCT reveals underlying morphologic alterations Bonn, Germany. that are detected by specific topographic fundus imaging mo- Supported by Grant O-137.0011 from the BONFOR Program (Fac- dalities. ulty of Medicine, University of Bonn); European Union Grant FP6, Integrated Project EVIGENORET Grant LSHG-CT-2005-512036; and the Kroener Foundation (Germering, Germany). METHODS Submitted for publication February 9, 2009; revised April 9, 2009; accepted August 7, 2009. Fifty-six eyes of 28 patients (8 male and 20 female patients) with Disclosure: P. Charbel Issa, Heidelberg Engineering (F); R.P. fundus abnormalities due to PXE were investigated. All patients were Finger, Heidelberg Engineering (F); F.G. Holz, Heidelberg Engineer- seen in the outpatient clinic at the Department of Ophthalmology, ing (F, C, R); H.P.N. Scholl, Heidelberg Engineering (F) University of Bonn, which is a tertiary PXE-referral center in Germany. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- The diagnosis of PXE was positively confirmed by a skin biopsy, ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. genetic analysis, or characteristic funduscopic pathologic changes in Corresponding author: Hendrik P. N. Scholl, Department of Oph- combination with further systemic manifestations typical for PXE. thalmology, University of Bonn, Ernst-Abbe-Strasse 2, D-53127 Bonn, All patients underwent a complete ophthalmic examination, includ- Germany, [email protected]. ing best corrected visual acuity, indirect ophthalmoscopy, fundus pho-

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FIGURE 1. Multiple examples for breaks in Bruch’s membrane in patients with PXE. Vertical arrows: small breaks; inclined arrows: larger breaks. The RPE epithelium overlying Bruch’s membrane may appear virtually normal, altered, focally detached, or absent. After growing from the choroidal space through the gaps in Bruch’s membrane, ﬁbrous tissue may be present underneath the neurosensory retina. Asterisk: outer nuclear layer. Arrowhead: junction between outer and inner photoreceptor segments.

tography (FF450; Carl Zeiss Meditec, Jena, Germany), and combined Engineering). The OCT B-scan is then repositioned in the moving eye cSLO and SD-OCT imaging (Spectralis HRA-OCT, Heidelberg Engineer- and thus stabilized and frozen at the selected retinal location. The ing, Heidelberg, Germany). software computes and compensates for movements between single The study was in compliance with the tenets of the Declaration of B-scan images caused by eye movements. Averaging live B-scans im- Helsinki, and informed consent was obtained from every patient. proves the signal-to-noise ratio and therefore enhances image quality with increased B-scan contrast and detail. Fundus Imaging The cSLO unit of the Spectralis HRA-OCT is similar to the widely used HRA2 (Heidelberg Engineering, Heidelberg, Germany) and uses an RESULTS optically pumped solid state laser source to generate the blue light excitation wavelength of 488 nm for FA and FAF images. Recorded Angioid Streaks emission wavelengths are limited by a barrier filter to wavelengths between 500 and 700 nm. A diode laser source of 820 nm wavelength On SD-OCT scans, the pathology of angioid streaks was local- is used for NIR reflectance recordings. With confocal image acquisi- ized to the Bruch’s membrane-retinal pigment epithelium tion, light from a conjugate plane of interest is detected by the image (RPE) complex (Fig. 1). The width of the gap in Bruch’s sensor, permitting suppression of light from planes anterior and pos- membrane was variable and the two breaking edges were terior to the plane of interest and resulting in high-contrast fundus usually in one horizontal plane (especially when gaps were images. narrow), but could also be displaced. The RPE overlying breaks The high-resolution SD-OCT has a 7-␮m optical depth resolution in Bruch’s membrane could seem unaffected, but often ap- and a 14-␮m lateral optical resolution. The system acquires 40,000 peared altered, focally detached, or absent. If a multitude of A-scans per second. In the present study, the B-scan angle was set to breaks was present on a single OCT scan, Bruch’s membrane 30° with 768 A-scans per B-scan, resulting in a lateral resolution of 11 appeared degraded into small pieces. In many advanced cases, ␮m/pixel and a scan rate of 50 B-scans per second. fibrovascular tissue extended from the choroidal into the sub- Using automated eye tracking and image alignment based on cSLO neurosensory space through the gaps in Bruch’s membrane. images, the software allows averaging a variable number of single Figure 2 shows that particularly in areas with retinal atrophy, images in real time (ART, [Automatic Real Time] Module; Heidelberg the gaps in Bruch’s membrane could be large. The breaks in

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such as accompanying RPE atrophy or hyperplasia. If viable RPE was present surrounding a streak visible on FAF images, contrast compared with background was usually better than in the FA or NIR reflectance images. Peau d’Orange With the cSLO, visualization of peau d’orange was best using confocal NIR reflectance imaging (Figs. 4, 5). On confocal NIR reflectance, peau d’orange appears with small flecks of lower reflectivity compared with background which can be conflu- ent, predominantly at the temporal posterior pole. FAF as well as FA were usually unremarkable or rarely revealed a fine mottling within areas of marked peau d’orange. Compared to flecks of lower reflectivity, areas of higher reflectivity on NIR reflectance showed an increased signal at the outer Bruch’s FIGURE 2. Fundus photograph (A) and near-infrared reflectance image membrane–RPE complex on SD-OCT scans (Fig. 5). (B) of a patient with PXE showing marked atrophic alterations. Breaks in Bruch’s membrane were only visible on the OCT scan (C). Peripheral Chorioretinal Atrophy Chorioretinal atrophic spots usually occur in the midperiphery this eye were not visible on funduscopy or NIR reflectance and were detected by all imaging modes used in this study. imaging. They showed increased reflectance on NIR images and an Comparison of images recorded by confocal NIR reflec- increased signal on FAF and late-phase FA. This increased signal tance, FAF, and FA revealed the most reliable detection of could be blocked by focal hyperpigmentations. Frequently, the angioid streaks in the NIR mode (Fig. 3). Breaks in the Bruch’s comet’s tail also showed an increased FAF signal. Since early membrane–RPE complex were usually present on SD-OCT angiographic sequences were always obtained from the poste- scans, even if angioid streaks were visible only on NIR reflec- rior pole (30° ϫ 30°), we do not know the appearance of tance but not on FAF imaging or FA (Figs. 3A–H). In some salmon spots in the early angiographic phase. cases, the break appeared to be restricted to the outer part of SD-OCT scans through lesions at the peripheral fundus are the Bruch’s membrane–RPE complex. The width of the angioid difficult to record. Therefore, only a few scans through salmon streak on NIR reflectance imaging corresponded to the defect spots are available. These showed hyporeflective spaces involv- in Bruch’s membrane visible on OCT imaging. If visible on FAF ing the outer neurosensory retina with a slightly hyperreflec- and FA, the width of the streaks could vary in those imaging tive inner lining and focal debris-like deposits just above the modalities depending on concomitant structural alterations, RPE level (Fig. 6).

FIGURE 3. Angioid streaks in PXE may be visible on the NIR reflectance (A; black arrows) but not on FAF (B)orFA(C). SD-OCT reveals breaks in Bruch’s membrane exactly corresponding to the angioid streaks visible on NIR reflectance imaging. Below the SD-OCT scans are magnified cutouts of the area marked by the white bars in (A–C) and by the black squares in (D). The RPE appears to cover the break in Bruch’s membrane below. (E–H) Similar findings in a larger streak that passes through the central posterior pole. (I–L) Streaks that are also clearly visible on FAF and FA imaging. On SD-OCT scans (L), the breaks appeared larger or with a small pigment epithelial detachment. Asterisk and arrowhead are as in Figure 1.

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FIGURE 4. Peau d’orange in PXE was not visible on fundus autofluorescence (A)orFA(B) but was easily detected on confocal NIR reflectance imaging (C, D). (D) Magnification of the black rectangle in (C).

Pattern Dystrophy-like RPE Changes discussing physical–optical phenomena, Kofler17 was the first to suggest this anatomic context. We used topographic In two patients with fundus findings similar to pattern dystro- cSLO imaging for detection of angioid streaks and simulta- phy, areas with an increased autofluorescence signal were neous SD-OCT imaging for quasi in vivo histologic assess- associated with material deposited below the neurosensory retina (Fig. 7). It was not possible to distinguish whether this ment. This study provides the first direct evidence that material was located within the RPE layer, which may be breaks in Bruch’s membrane are indeed the underlying pa- focally distended, or just below or above the RPE. thology of angioid streaks. The good visibility of the broken edges on SD-OCT scans may be explained by the consider- able thickening and calcification of Bruch’s membrane in DISCUSSION patients with PXE.7,8 On confocal NIR reflectance imaging, breaks in Bruch’s It is known from histopathologic studies that the primary alteration at the fundus in PXE is located in Bruch’s mem- membrane appear darker compared with the surrounding fun- brane.7–12 In our study, confocal NIR reflectance imaging at dus reflex. Similarly, anatomic structures with interruption of Bruch’s membrane, such as at the optic disc rim, show a 820 nm turns out to be an excellent noninvasive method for 16 imaging such abnormalities in Bruch’s membrane in vivo. El- decrease in reflectance. This decrease suggests that Bruch’s sner et al.16 were the first to suggest this imaging technique for membrane is a significant reflector for NIR light at the fundus, visualizing deep retinal and subretinal structures. In retinal and it may even be increased in patients with PXE due to the imaging, confocality enables visualization of alterations of a calcification of Bruch’s membrane. The superior visibility of given plane with improved contrast and detail. Scattered light peau d’orange with confocal NIR reflectance also locates the is not detected by the cSLO but leads to decreased image primary underlying disease below the RPE. Assuming that cal- quality when a fundus camera is used. Therefore, cSLO NIR cification of Bruch’s membrane further increases the reflectiv- reflectance fundus images are mainly affected by reflection and ity on NIR reflection, the darker flecks in peau d’orange would absorption. Moreover, melanin only mildly absorbs NIR light, represent areas still spared from calcification. The herein pre- leading to good visibility of structures below the RPE and to sented findings on SD-OCT scans (Fig. 5) support this hypoth- less interindividual variation depending on overall fundus pig- esis. The neurosensory retina overlying peau d’orange revealed mentation.16 no characteristic pathologic formations on SD-OCT scans. Ac- In histopathologic studies, eyes of patients with PXE cordingly, retinal sensitivity and dark adaption characteristics revealed the common finding of breaks in Bruch’s mem- were reported not to be affected in areas of peau d’orange.18 brane with a topographic arrangement consistent with that The poorer detection of angioid streaks on FAF and FA of angioid streaks.7–12 Based on clinical observations and imaging may be explained by the stronger absorbance of

FIGURE 5. Alterations of peau d’orange on SD-OCT. The black rect- angles in (A) and (E) mark the area that is enlarged in (B) and (F), where the black lines indicate the place- ment of the OCT scans (C, G). The magnified sector of the OCT scan reveals that areas with higher reflectivity on the confocal NIR images in (B) and (F) show increased reflectivity within the outer zone of RPE-Bruch’s membrane complex. The arrows in (F) and (H) mark the same section, revealing that the dark spots in peau d’orange show a decreased reflectivity in the outer RPE–Bruch’s membrane complex. The images suggest that calcification of Bruch’s membrane leads to increased reflectivity on near infrared as well as on OCT imaging.

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monochromatic light of 488 nm by melanin and lipofuscin granules in the RPE, which can be preserved overlying angioid streaks. Therefore, on FAF and FA images, angioid streaks may only become visible with concomitant alterations of the RPE, such as loss of pigment granules or ruptures in the cell layer as described in histologic studies.7,8 RPE damage may then extend beyond the lateral margins of breaks in Bruch’s membrane. Further studies are necessary to assess whether these alterations of the RPE layer may have functional (e.g., localized scotomas) and/or prognostic (e.g., on the development of choroidal neovascularization) consequences. SD-OCT may serve as a tool to investigate PXE-related abnormalities. For instance, subneurosensory fibrous tissue and breaks in Bruch’s membrane in areas of retinal atrophy were not consistently identified on cSLO images or fundus photography, but were detected by SD-OCT. Dislocation of fragmented pieces of Bruch’s membrane from a common plane was previously interpreted as fixation artifacts in histologic studies.7 However, in vivo SD-OCT revealed that such displacements of Bruch’s membrane fragments are a real feature of PXE. In patients with very advanced fundus changes such as large areas of atrophy and fibrotic alterations, the underlying primary pathologic characteristic— breaks in a thickened and degenerated Bruch’s membrane— cannot be detected on FA, fundus photography, NIR, or FAF imaging.5 Hu et al.1 stated that RPE atrophy may “become so extensive around the disc and in the macular area that one can no longer see any signs of angioid streaks” and that this may complicate the ophthalmic diagnosis of PXE. Noninva- sive SD-OCT imaging may still detect the characteristic breaks in Bruch’s membrane and therefore allows uncover- ing the pathogenic context. Pattern dystrophy–like changes have been reported to be associated with PXE.4,19 A histologic study of an eye with pattern dystrophy due to a mutation in the RDS/peripherin gene revealed a thickened Bruch’s membrane, a greatly distended RPE due to excessive accumulation of lipofuscin 20 and partially atrophic photoreceptors. SD-OCT imaging in FIGURE 7. Pattern dystrophy-like fundus changes in a patient with a patient with PXE with apparent pattern dystrophy-like PXE. Areas with an increased autofluorescence signal are associated changes revealed similar findings: a thickened Bruch’s mem- with deposit-like alterations below the neurosensory retina. The outer brane–RPE complex with corresponding increased FAF sig- nuclear layer overlying these lesions was decreased in thickness. nal indicating accumulation of autofluorescent material (Fig. 7). In PXE, a generalized retinal and RPE alteration due to the abnormal underlying Bruch’s membrane have been sug- mal findings on electro-oculography despite normal neuro- gested,21 and pattern dystrophies may present with subnor- sensory function, indicating a diffuse dysfunction of the RPE. We suggest that pattern dystrophy-like changes in PXE may rather represent a phenotypic characteristic shared by diseases with alterations of the Bruch’s membrane–RPE layer complex. In conclusion, multimodal cSLO imaging in combination with SD-OCT appears to be an excellent imaging method for PXE-related ocular disease. Since simultaneous SD-OCT and multimodal cSLO imaging are capable of detailed investigation of the Bruch’s membrane–RPE photoreceptor complex, it appears to be a promising technology for the study of other diseases that primarily affect this anatomic complex, such as age-related macular degeneration.15,22,23

References

FIGURE 6. SD-OCT scans through peripheral chorioretinal atrophic 1. Hu X, Plomp AS, van SS, Wijnholds J, de Jong PT, Bergen AA. spots in two patients with PXE. (A, B) FAF shows an increased signal Pseudoxanthoma elasticum: a clinical, histopathological, and mo- within the area of the atrophy, presumably originating from the sclera. lecular update. Surv Ophthalmol. 2003;48:424–438. OCT scans (C, D) revealed hyporeﬂective spaces of the outer neuro- 2. Finger RP, Charbel Issa P, Ladewig MS, et al. Pseudoxanthoma sensory retina with a slightly hyperreﬂective inner lining and focal elasticum: genetics, clinical manifestations and therapeutic ap- debris-like deposits just above the RPE-level. proaches. Surv Ophthalmol. 2009;54:272–285.

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3. Chassaing N, Martin L, Calvas P, Le BM, Hovnanian A. Pseudoxan- 14. Wojtkowski M, Srinivasan V, Fujimoto JG, et al. Three-dimensional thoma elasticum: a clinical, pathophysiological and genetic update retinal imaging with high-speed ultrahigh-resolution optical coher- including 11 novel ABCC6 mutations. J Med Genet. 2005;42:881– ence tomography. Ophthalmology. 2005;112:1734–1746. 892. 15. Fleckenstein M, Charbel Issa P, Helb HM, et al. High resolution 4. Agarwal A, Patel P, Adkins T, Gass JD. Spectrum of pattern dystro- spectral domain-OCT imaging in geographic atrophy associated phy in pseudoxanthoma elasticum. Arch Ophthalmol. 2005;123: with age-related macular degeneration. Invest Ophthalmol Vis Sci. 923–928. 2008;49:4137–4144. 5. Sawa M, Ober MD, Freund KB, Spaide RF. Fundus autofluores- 16. Elsner AE, Burns SA, Weiter JJ, Delori FC. Infrared imaging of cence in patients with pseudoxanthoma elasticum. Ophthalmol- sub-retinal structures in the human ocular fundus. Vision Res. ogy. 2006;113:814–820. 1996;36:191–205. 6. Shiraki K, Kohno T, Moriwaki M, Yanagihara N. Fundus autofluo- 17. Kofler A. Beitra¨ge zur Kenntnis der Angioid streaks. Arch Augen- rescence in patients with pseudoxanthoma elasticum. Int Ophthal- heilkd. 1917;82:134–149. mol. 2001;24:243–248. 18. Holz FG, Jubb C, Fitzke FW, Bird AC, Pope FM. Dark adaptation 7. Bo¨ck J. Zur Klinik und Anatomie der gefa¨ssa¨hnlichen Streifen im and scotopic perimetry over ‘peau d’orange’ in pseudoxanthoma Augenhintergrund. Z Augenheilkd. 1938;95:1–50. elasticum. Br J Ophthalmol. 1994;78:79–80. 8. Hagedoorn A. Angioid streaks. Arch Ophthalmol. 1939;21:746– 19. McDonald HR, Schatz H, Aaberg TM. Reticular-like pigmentary 774. patterns in pseudoxanthoma elasticum. Ophthalmology. 1988;95: 9. Verhoeff FH. Histological findings in a case of angioid streaks. Br J 306–311. Ophthalmol. 1948;32:531–544. 20. Zhang K, Garibaldi DC, Li Y, Green WR, Zack DJ. Butterfly-shaped 10. McWilliam RJ. On the histology of angioid streaks. Trans Ophthal- pattern dystrophy: a genetic, clinical, and histopathological report. mol Soc U K. 1951;71:243–249. Arch Ophthalmol. 2002;120:485–490. 11. Jensen OA. Bruch’s membrane in pseudoxanthoma elasticum. His- 21. Audo I, Vanakker OM, Smith A, et al. Pseudoxanthoma elasticum tochemical, ultrastructural, and x-ray microanalytical study of the with generalized retinal dysfunction, a common finding? Invest membrane and angioid streak areas. Albrecht Von Graefes Arch Ophthalmol Vis Sci. 2007;48:4250–4256. Klin Exp Ophthalmol. 1977;203:311–320. 22. Wolf-Schnurrbusch UE, Enzmann V, Brinkmann CK, Wolf S. Mor- 12. Dreyer R, Green WR. The pathology of angioid streaks: a study of phologic changes in patients with geographic atrophy assessed twenty-one cases. Trans Pa Acad Ophthalmol Otolaryngol. 1978; with a novel spectral OCT-SLO combination. Invest Ophthalmol 31:158–167. Vis Sci. 2008;49:3095–3099. 13. Srinivasan VJ, Wojtkowski M, Witkin AJ, et al. High-definition and 23. de Bruin DM, Burnes DL, Loewenstein J, et al. In vivo three- 3-dimensional imaging of macular pathologies with high-speed dimensional imaging of neovascular age-related macular degener- ultrahigh-resolution optical coherence tomography. Ophthalmol- ation using optical frequency domain imaging at 1050 nm. Invest ogy. 2006;113:2054–e1–14. Ophthalmol Vis Sci. 2008;49:4545–4552.

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