Genetic and environmental factors

Large-scale studies have implicated genetic risk variants of the ARMS2 gene and polymorphisms in complement genes including factor H with AMD.13 ,14–17 Interestingly, an association between RPD and the ARMS2 gene variants was demonstrated in Asian and Caucasian populations of AMD patients.13 ,18 ,19 Puche and coworkers evaluated four genes associated with AMD (CFH, ARMS2/HTRA1, C3, apolipoprotein E) as well as environmental factors in AMD patients with RPD compared with AMD patients without RPD. In accordance with other studies, neither the environmental factors nor the genotypes studied were significantly associated with either group. Both groups shared the same major genetic and environmental factors and showed no significant differences suggesting that RPD occur in the same genotype and epidemiological background as AMD.20 Boddu et al examined the genotypes of distinctly selected patients with AMD and RPD and no evidence of soft drusen and compared them with AMD patients with large soft drusen and no RPD. In their study, there were no significant differences found in the distribution of the ARMS2 risk allele and the CHF risk allele between both groups. Yet, a significant association was found with increased age, later age of AMD onset, female gender and risk for systemic hypertension.21 Further advanced genetic studies must verify the exact correlation between RPD and AMD risk alleles as well as an association between RPD pathophysiology and other genes.

Pathophysiology

The pathophysiological mechanisms underlying the formation of RPD lesions are unknown. Based on en face OCT imaging, Sohrab and coworkers attributed the pathognomic pattern of RPD to physically correlating structures within the choroidal stroma and the choroidal vasculature and interpreted subretinal deposits misleadingly as secondary pathological changes that do not consistently correlate with the RPD pattern.22 Other studies support the hypothesis that RPD may be anatomically related to the underlying choroidal vasculature.23 By contrast, Vongkulsiri et al found no concordance between RPD and large choroidal vessels using stereological analysis of SD-OCT images and, therefore, recommend abandoning the hypotheses postulating anatomic correlations between RPD and large choroidal vessels.24

Spaide, Curcio and coworkers showed on histological examination and multimodal imaging that RPD are located internal to the retinal pigment epithelium (RPE) and hypothesise that the biological substrate of RPD is generated at the level of the RPE and photoreceptor outer segments.25 ,26

In a recent study, our group demonstrated that the localisation of evolving RPD seems to be related to the presence and site of choroidal watershed zones, suggesting that choroidal hypoxia may play a role in RPD pathogenesis (figure 1I).27 In addition to other authors who reported a reduced choroidal thickness in eyes with RPD, we also showed a decreased choroidal volume in eyes with evolving RPD.27–30 Eventually, a reduction in macular choroidal volume in patients with RPD was confirmed using 3D-1060 nm OCT maps as well as swept-source OCT technology.31 ,32 Furthermore, en face images through the choroid showed predominantly narrow and sparse choroidal vessels in these patients. However, all these observations may be interpreted as either cause or consequence of RPD development in the subretinal space (figure 1F). Besides, the relationship between RPD and choroidal thinning is presumably not a simple direct correlation, and both RPD and choroidal thinning may be related to other factors.33

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Figure 1

(A–I) Reticular pseudodrusen (RPD) shown in different imaging modalities. First row: (A) Colour fundus photography displays RPD as yellowish-pale lesions that have a more punctate appearance closer to the fovea. (B) Confocal scanning laser ophthalmoscopy (cSLO) near infrared reflectance (IR) showing hyporeflective lesions. (C) Multi-spectral cSLO combines blue reflectance, green reflectance and IR in one image and depicts RPD lesions as greyish-yellowish lesions. Note that the target aspect in the centre of RPD lesions is revealed more clearly and distinctly than in other modalities. (A–C) Arrowheads mark an exemplary RPD lesion exhibiting a central target aspect within the lesion. Asterisks demonstrate an area affected by RPD lesions lacking central targets. Second row: (D) Spectral-domain optical coherence tomography (SD-OCT) scans showing change of lesion height at baseline (above) and at 12-month follow-up (below). Arrows point to retinal vessel crossings proving a good alignment between baseline and follow-up scan. Asterisks mark a lesion developing from stage 2 to 3. Arrowheads point to a lesion that appears not to have changed and stars mark a lesion that progressed from stage 1 to 2. (E) Magnified cut-out of a cSLO fundus autofluorescence (FAF) image of the patient seen in first row. Progression of RPD affected retinal area is clearly demonstrated temporal superior to the fovea at the border of the RPD affected area between baseline (above) and 12-month follow-up (below). Ovals indicate region of growth in RPD affected area suggesting a centrifugal spread. The lesions’ density appears lower at the border of the affected area. (F) Choroidal volume map with the Early Treatment Diabetic Retinopathy Study grid of an eye with RPD obtained by enhance depth imaging SD-OCT volume scans. Notably, mean choroidal thicknesses and choroidal volumes decrease from baseline (left) to 12-month follow-up (right). Third row: cSLO FAF image of a patient with a multilobular geographic atrophy at baseline (G) and at 2-year follow-up (H). Note the growth dynamics of atrophy lobes and hyperautofluorescent lesion margins towards the previously RPD-affected area. (I) Indocyanine green video-angiography (ICG) during early phase in a patient with beginning RPD shows a clearly demarked early hypofluorescence representing a stellate choroidal watershed zone (CWZ). Magenta arrowheads indicate CWZ border. RPD area is localised within the CWZ in this patient, yet, cannot be detected in the early ICG phase.

Spaide recently presented data on a long-term clinical course of eyes with RPD showing regression of lesions accompanied by a developing outer retinal atrophy and a loss of the underlying choroidal thickness. The author proposes the term ‘outer retinal atrophy’ as a new entity in late stage AMD. The author's theory on the biogenesis of RPD focuses on a dysfunction of the RPE. A reduction in RPE function may lead to dysfunctional transport mechanisms between the RPE and the Müller cells resulting in an accumulation of material in the outer retina. This material may impede normal transportation of outer segments toward the RPE going along with a thinning of the outer retina and the underlying choroid. With attenuation of the photoreceptor metabolic activity, less oxygen is needed resulting in a choroidal thinning.34 Immunological and genetic studies suggest that the complement system plays an essential role in the pathophysiology of AMD. Local effects, such as impairment of complement activation control at the level of the RPE, may possibly contribute to the accumulation of RPD in the subretinal space.35

Interestingly, Marsiglia and colleagues recently reported that geographic atrophy (GA) expanded particularly into areas previously affected by RPD (figure 1G,H). Based on these results, the authors postulate that RPD represent an early manifestation of the process leading to GA.36 Both AMD phenotypes may underlie similar pathophysiological mechanisms. The fast progressing ‘diffuse-trickling’ GA subtype shows a strikingly higher incidence of RPD compared with the other GA subtypes, which additionally supports this hypothesis.37

A recent study based on SD-OCT and cSLO infrared reflectance (IR) imaging found that RPD represents an independent risk factor for GA development, but not choroidal neovascularisation (CNV) development, in the fellow eye of patients being treated with antivascular endothelial growth factor therapy for CNV in the first eye.38 Hogg and colleagues reported that patients with unilateral neovascular AMD and RPD present in their fellow eyes are at significantly higher risk of progression to bilateral disease. Thus, the authors recommend careful monitoring of such patients due to the risk of developing bilateral neovascular disease.39

Histology

In 1988, Sarks and coworkers showed that components of soft drusen and basal linear deposit are also found in vacuoles within the RPE, basal mounds within basal laminar deposit and within the subretinal space.40 The histological correlate that presumably corresponds to the appearance of RPD in various imaging modalities was later called SDD (figure 2).25 The question of the exact histopathological correlate of RPD remains not fully solved and is still a matter of ongoing debate. The shortage of histopathological material of eyes with RPD previously imaged by current high resolution imaging is a limiting factor. Sarks et al41 showed in one clinicopathological examination that RPD may be located in the subretinal space on the internal surface of the RPE. Interestingly, immunohistochemistry and confocal microscopy revealed that molecules that are also typically associated with soft drusen, such as unesterified cholesterol, apolipoprotein E, complement factor H and vitronectin, were also found in SDD.42 ,43 A lack of opsins suggests that the material was not derived from photoreceptor outer segments. SDD are hypoautofluorescent at wavelengths used in fundus autofluorescence (FAF) imaging, and so SDD are presumably not directly derived from outer segments that are rich in retinoids. The 2-lesion, 2-compartment hypothesis was firstly elaborated by Curcio et al in 2013 and is based on the idea that many of the factors present in the sub-RPE space for drusen biogenesis are also present in the subretinal space including lipid transport. In the 2-lesion, 2-compartment model, lipid recycling and bidirectional lipoprotein secretion by polarised RPE are unifying factors.26 RPD, histologically seen as SDD, are rich in unesterified cholesterol and poor in esterified cholesterol unlike soft drusen setting apart SDD as a lesion of distinct composition.44

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Figure 2

(A–B) Reticular pseudodrusen (RPD) histologically seen as subretinal drusenoid deposit (SDD). (A) A 0.8-mm-thick epoxy resin section, toluidine blue stain; 80-year-old man. This foveal centre has a dysmorphic retinal pigment epithelium (RPE), basal laminar deposits, basal mounds and three discrete SDD formations. Photoreceptor morphology is disturbed over all formations, manifest as outer segment (OS) shortening (1 and 3), and OS loss with inner segment deflection and absence (2). Bar=50 µm. (B) At 1.8 mm nasal to the foveola, individual SDD formations are dollop shaped and as small as a single RPE cell. SDD contains vesicular components, and septa are apparent (arrows). OS of overlying photoreceptors are closely opposed to SDD internal surface. Bar=20 µm. IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; FH, Henle fibre layer; IPL, inner plexiform layer. Courtesy of CA Curcio, University of Alabama at Birmingham.26

Curcio et al recently found in a histological study that SDD are associated with photoreceptor degeneration, including deflected, shortened, and missing inner and outer segments (figure 2). SDD is a prevalent finding comparably with basal linear deposit and located preferentially in the perifovea, whereas basal linear deposits were predominantly found in the fovea.26 Based on these findings, the authors hypothesised that this distinct topography reflects the different nature of rod and cone photoreceptor physiology.

Imaging

In the year 2010, studies using combined cSLO and SD-OCT revealed the localisation of RPD lesions internal to the RPE.3 ,5 ,25 Subsequently, Zweifel et al and Querques et al identified different stages of RPD describing the varying degree of accumulation of extracellular material (figure 1D). Stage 1 is defined as diffuse deposition of granular hyperreflective material between the RPE and the ellipsoid zone (EZ).45 Stage 2 is characterised as mounds of accumulated material sufficient to alter the contour of the EZ. Stage 3 is defined by thicker material that adopted a conical appearance and broke through the EZ. Stage 4 is reached when material fades and migrates within the inner retinal layers.46 ,47

Several multimodal imaging studies showed that RPD are most prevalent in the superior macula and that FAF, IR and SD-OCT are superior to other modalities in detecting RPD including fundus photography, blue channel image of fundus photography, near-infrared FAF, confocal blue reflectance, fluorescein angiography and indocyanine green angiography.46–51 Several authors advise the use of at least two imaging modalities for accurate RPD identification. All studies consistently report a high degree of bilaterality, a preponderance of the female sex and the occurrence of RPD in all AMD phenotypes such as GA, CNV and drusen maculopathy, yet showing the highest prevalence of RPD in multilobular GA.4 ,7 ,10 ,13 ,41 ,46 ,48–53

In a retrospective study by Suzuki et al, three different types of RPD were identified: dot and ribbon RPD in the macular area and peripheral RPD each presenting as subretinal deposits on SD-OCT yet showing different imaging properties in colour fundus photographs and infrared images. This raises the question of different biochemical compositions in each RPD type as well as pathophysiological dissimilarities possibly resulting in different risks of progression to late stage AMD.54

Querques and coworkers reported on a progression of RPD lesions further into the photoreceptor layers over a 2-year follow-up period and Steinberg and coworkers showed in another longitudinal study that the retinal area affected by RPD lesions grows at 4.4 mm² per year in patients with GA (figure 1D, E).47 ,55

Spaide investigated the long-term clinical course of eyes with RPD and evaluated the photoreceptor length and the choroidal thickness at baseline and at follow-up visits in eyes showing complete regression of RPD lesions. Over a mean of 2.9-year follow-up period, the underlying choroidal thickness decreased to 81.4% of its initial value and the photoreceptor length to 74.4% of the initial length (figure 1F).34 Those three studies underline the dynamic nature of RPD lesions over time. Auge et al confirm such dynamic processes in the development and changes of RPD over time and, yet, emphasise the importance of exact registration of SD-OCT B-scans at different time points as well as the use of very dense volume scans in order to be capable of reliably assessing such discrete intraretinal changes over time.56

A distinct, frequently prevalent feature of RPD lesions is the so-called ‘target aspect’, which was first described by Querques et al in 2012 referring to a certain appearance of some RPD lesions with a hyporeflective annulus and an isoreflective centre in cSLO IR imaging (figure 1B).28 The authors attribute the target aspect to an interruption of the EZ, which correlates to RPD stage 3 according to the classification by Zweifel et al and Querques et al.46 ,47 Recently, our group demonstrated that identification of RPD target aspects can be improved by multispectral cSLO imaging (figure 1C).57

Using AO, Mrejen et al investigated the cone photoreceptor mosaic in eyes with RPD and compared the cone density with eyes with soft drusen. Interestingly, they report a dramatic reduction in cone density over RPD lesions possibly due to a change in their orientation, an alteration of their cellular architecture or even absence of the cones themselves. This suggests that eyes with RPD may experience decreased retinal function independent of the presence of CNV or GA and confirms findings in recent functional studies.58 In AO en face imaging, soft drusen appear as highly hyper-reflective lesions, centred and/or surrounded by a hyporeflectivity, whereas RPD appear as isoreflective lesions, surrounded by a hyporeflective halo.59 Defined stages of RPD could be correlated to reflectivity changes in AO-SLO consistent with perturbed surrounding photoreceptors. AO-SLO imaging in conjunction with OCT suggested that the hyporeflective annulus likely consists of photoreceptors with deflected, degenerated, or missing inner or outer segments according to histology.26 ,60 The hyporeflective annulus seen by AO-SLO corresponds to a hyporeflective gap in the EZ on either side of the central core as visible in SD-OCT.60 Using AO-SLO and AO-OCT, Meadway and coworkers recently demonstrated that the central reflective core of RPD lesions consists of RPD material itself and not photoreceptor material.61

Structure-function analysis

Efforts have been made to answer the question whether structural changes associated with RPD lesions have an impact on retinal function. Contrary to other phenotypic characteristics of AMD, our group reported that multifocal electroretinography (mfERG) measurements do not show definite interference of electrophysiological activity in retinal areas affected exclusively with RPD representing the first structure-function analysis of RPD.62 Yet, mfERG allows for measuring cone function only. In another structure-function analysis based on microperimetry, Querques and colleagues reported a greater extent of reduced sensitivity in eyes with RPD compared with eyes with typical soft drusen.63 Microperimetry after dark adaptation allows investigating both cone and rod activity, which may explain discrepancies between both series as the latter study showed a reduced sensitivity. This may be in line with the theory of Curcio and coworkers that RPD is mainly derived from rod physiology.26 ,64 Studies on RPD potentially causing a rod-mediated dark adaptation impairment would be of interest in this context.65 ,66 Ooto and coworkers found that distribution and number of RPD lesions are closely associated with retinal sensitivity in microperimetry measurements.67 In accordance, Forte and colleagues confirmed those results by measuring a reduced sensitivity in microperimetry despite preserved visual acuity in RPD patients.68 In a longitudinal structure-function analysis, our group recently reported that mfERG allows for detecting a decline of function over 1 year in eyes with progressive RPD. Yet, functional decline could not be correlated to changes in individual morphological parameters such as RPD number or RPD affected area size.69 Hogg et al39 found a reduced near visual acuity and an impaired spatial vision under conditions of reduced contrast and luminance in RPD eyes.

Perspective

RPD represent an important component of AMD pathology localising to the subretinal space. RPD are clinically defined by their imaging properties and histologically by their localisation and biochemical composition. RPD detection based on multimodal imaging should systematically be part of AMD screening examinations.

Due to the small lesion size, their confluent reticular pattern and frequently due to a spread beyond the central macula, quantification of RPD in terms of total affected area as well as extent in height is much more challenging compared with quantification of other AMD phenotypes like soft drusen, GA or CNV.55 ,70 Nevertheless, a standard imaging processing software instrument needs to be established for automated and consistent evaluation of RPD status in the future.

Most former natural history studies as well as interventional studies in early AMD did not include imaging RPD beyond colour fundus photography. In the light of growing importance of RPD, this phenotype must be included in all study protocols of upcoming natural history and interventional AMD trials for learning more about longitudinal evolution of RPD as well as their response to therapy. Most importantly, identifying the molecular substrate and the biogenesis of RPD lesions will open the door to identifying further therapeutic targets in AMD treatment.