Multimodal Imaging in Choroideremia

Abstract

Choroideremia (CHM) is associated with progressive degeneration of the retinal pigment epithelium (RPE), choriocapillaris (CC), and photoreceptors. As animal models of CHM are lacking, most information about cell survival has come from imaging affected patients. This chapter discusses a combination of imaging techniques, including fundus-guided microperimetry, confocal and non-confocal adaptive optics scanning laser ophthalmoscopy (AOSLO), fundus autofluorescence (FAF), and swept-source optical coherence tomography angiography (SS-OCTA) to analyze macular sensitivity, cone photoreceptor outer and inner segment structure, RPE structure, and CC perfusion, respectively. Combined imaging modalities such as those described here can provide sensitive measures of monitoring retinal structure and function in patients with CHM.

23.1. Introduction

Choroideremia (CHM), which is estimated to affect 1:50,000, is an X-linked recessive disease caused by a mutation in the CHM (REP1) gene on chromosome Xq21 (Aleman et al. 2017). CHM leads to degeneration of the choriocapillaris (CC), retinal pigment epithelium (RPE), and the photoreceptors. Patients develop progressive loss of night vision, subsequent peripheral visual field loss, and eventual central vision loss. The pathogenetic mechanism underlying the degeneration is not clearly understood but may be due to a deficiency in the function of proteins which have a role in organelle formation and trafficking of vesicles (Coussa and Traboulsi 2012).

The order that the retinal layers are affected by degeneration in patients with CHM is not clear. A study using fundus autofluorescence (FAF) images, adaptive optics scanning laser ophthalmoscopy (AOSLO), and spectral-domain optical coherence tomography (SD-OCT) found that early degeneration of RPE cells likely occurs simultaneously with degeneration of photoreceptors (Syed et al. 2013). Studies using a combination of SD-OCT and confocal and non-confocal split-detector AOSLO techniques (Sun et al. 2016) and a study using OCT angiography (OCTA) imaging (Jain et al. 2016) concluded that RPE degenerates before photoreceptors. Another study performed AOSLO, OCT, and FAF imaging and found that RPE is the primary site of degeneration and also that photoreceptors may degenerate independently (Morgan et al. 2014). Other studies (Jacobson et al. 2006; Aleman et al. 2017) used OCT and psychophysical tests to demonstrate loss of photoreceptors first, perhaps independently or in conjunction with RPE depigmentation. A group using OCTA and FAF (Parodi et al. 2018) found that the CC maintained normal structure until RPE loss occurred.

23.2. Fundus-Guided Microperimetry

Fundus-guided microperimetry using Macular Integrity Assessment (MAIA, Centervue Inc., Fremont, CA) can be used to analyze macular sensitivity of patient eyes with CHM (Jolly et al. 2017). This instrument uses scanning laser ophthalmoscopy (SLO) with real-time fundus tracking at a rate of 25 frames/second using fundus landmarks as a reference for perimetry. It uses a superluminescent diode of 850 nm with 1024 × 1024 pixel resolution and a 36 × 36 degree field of view. Goldmann III (26 arcmin) stimuli are presented for 200 ms on a 1.27 cd/m² background with a dynamic range of 36 dB (Crossland et al. 2012; Dimopoulos et al. 2016).

23.3. Confocal and Split-Detector Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO)

AOSLO confocal imaging is an in vivo, noninvasive technique that records light emerging from the cone waveguide, which comprises scattered light from the IS/OS junction and the posterior tip of the outer segment. If both reflections are missing or are very weak, then the cone does not appear. AOSLO works by measuring higher order ocular aberrations via wavefront sensing and then compensates for these with a deformable mirror (Roorda et al. 2002). AOSLO has been used to visualize photoreceptors in eyes with retinal degeneration (Duncan et al. 2007; Roorda et al. 2007), including choroideremia (Syed et al. 2013; Sun et al. 2016; Morgan et al. 2018).

Photoreceptor inner segments have been visualized in vivo using non-confocal split-detector AOSLO (Scoles et al. 2014). This technique uses a reflective mask with an annulus in the image plane in place of a regular pinhole typically used for confocal detection. This method allows the confocal signal to be reflected into one detector and then directs the multiply scattered, non-confocal light from opposing sides of the annular aperture into two separate detectors. The split-detector signal is calculated as the difference between the two non-confocal detectors divided by their sum (Scoles et al. 2014). Non-confocal split-detector AOSLO can be especially useful in distinguishing areas where cone inner segments remain but outer segments are not waveguiding (Scoles et al. 2017).

23.4. Fundus Autofluorescence (FAF)

Photoreceptor outer segments and RPE cells naturally exhibit autofluorescence from bisretinoid constituents such as A2E which can be excited from an external light source and then imaged (Sparrow et al. 2012). Many FAF images are acquired using in vivo confocal scanning laser ophthalmoscopy (SLO) with short-wavelength (SW-AF) (488 nm) excitation and a 500 nm barrier filter to block reflected light and permit autofluorescent light from the fundus to pass through (Schmitz-Valckenberg 2008). SW-AF SLO imaging uses a confocal pinhole, which selectively allows imaging of a single plane to reduce noise from structures other than the retina (crystalline lens) that may contain fluorophores (Sparrow 2018). FAF can reveal lipofuscin fluorophores that amass in healthy normal and diseased RPE cells from pigment granules composed of lipid residues (Sparrow 2018). When excess lipofuscin accumulates in RPE cells due to incomplete photoreceptor outer segment degradation due to disease, it builds up and appears hyperfluorescent on FAF images (Schmitz-Valckenberg 2008).

Near-infrared autofluorescence (NIR-AF) excitation with a laser diode at 787 nm excitation and a barrier filter allowing light to pass at >810 nm can also be used to image fundus fluorophores, most likely derived from melanin pigment (Weinberger 2006). NIR-AF is more comfortable for patients and may pose less risk of RPE damage than SW-AF (Cideciyan et al. 2007; Cideciyan et al. 2015). NIR-reflectance (NIR-REF) imaging has been shown to be strongly correlated with NIR-AF imaging (Weinberger 2006) and uses similar wavelengths as are used to acquire infrared fundus images during OCT scans, which suggests that signals from OCT imaging might be comparable to NIR-REF and NIR-AF. Studies of patients with CHM quantified areas of preserved RPE and inner segment/outer segment (IS/OS) junction or inner segment ellipsoid zone (EZ) on FAF and OCT images and found that RPE degenerated prior to photoreceptors (Hariri et al. 2017) and found SW-AF to be repeatable over time in CHM patients (Jolly et al. 2016). A more recent study found differences in areas of preservation when comparing NIR-AF to short-wavelength autofluorescence (SW-AF) (Paavo et al. 2018).

23.5. Swept-Source Optical Coherence Tomography (SS-OCT)

OCT is a noninvasive, in vivo imaging technique used to image the fundus layers in cross section (Podoleanu and Rosen 2008; Huang et al. 2014). Swept-source OCT (PLEX Elite 9000, Carl Zeiss Meditec Inc., Dublin, CA) uses a 1060 nm tunable laser which can scan up to 100,000 A-scans/second with an axial resolution of 6.3 μm (Akman 2018). En face SS-OCT slabs have been used to assess geographic atrophy associated with age-related macular degeneration using large scans, up to 12 × 12 mm, comparable to 40 degree field of view (Thulliez et al. 2019). Unlike FAF which represents autofluorescent lipofuscin, when SS-OCT en face slabs are used to observe the RPE layer, the signal comes from RPE melanin (Greenstein et al. 2017). SS-OCT can be used to visualize RPE and semiautomatically identify borders of preserved RPE from en face slabs extending from the outer boundary of the outer plexiform layer (outer retina) to 8 μm beneath Bruch’s membrane (CC) (Zhang et al. 2017).

23.6. Swept-Source OCT Angiography (OCTA)

SS-OCTA can be used to visualize the CC in vivo and its associated flow voids (FV) (Zhang et al. 2018). CC perfusion can be measured as FV, defined as a percentage of the imaged region without measurable CC flow, using a threshold of one standard deviation below the mean CC flow from a normative database of 20 normal subjects aged 20–39 years old (Zhang et al. 2018). Prior studies of CHM using OCTA have suggested that the RPE area of loss was more extensive than the CC nonperfusion area, which in turn was larger than the area of retinal vascular nonperfusion (Jia et al. 2015). These results suggest RPE cells to be the primary site of degeneration, followed by loss of the CC and then photoreceptors. However, correlation of all the modalities discussed acquired concurrently should provide additional insight into the relationship between cellular function and structure in patients with CHM.

23.7. Conclusion

The mechanisms of degeneration in CHM remain unclear. While some studies using AOSLO suggest that RPE cells degenerate earliest (Morgan et al. 2014), others suggest RPE cells and photoreceptors degenerate independently (Syed et al. 2013), and structural measures may not demonstrate early changes in photoreceptor function (Jacobson et al. 2006; Aleman et al. 2017; Duncan et al. 2002). Recent advances in imaging technology permit assessment of eyes with CHM using multiple modalities to improve the study of these cells. The use of multimodal, noninvasive imaging may provide better understanding of the sequence of degeneration in eyes with CHM. Future studies are necessary to examine longitudinal data and degeneration using multimodal techniques, including those described here. While microperimetry can provide a measure of macular sensitivity, AOSLO can visualize photoreceptor morphology. FAF as well as SS-OCT can provide images of RPE structure, and SS-OCTA can display CC perfusion. Greater understanding of degeneration and disease progression is crucial to advance the development of novel therapies for this relentless, sight-threatening disease.