Microscopic inner retinal hyper-reflective phenotypes in retinal and neurologic disease

Abstract

Purpose: We surveyed inner retinal microscopic features in retinal and neurologic disease using a reflectance confocal adaptive optics scanning light ophthalmoscope (AOSLO).

Methods: Inner retinal images from 101 subjects affected by one of 38 retinal or neurologic conditions and 11 subjects with no known eye disease were examined for the presence of hyper-reflective features other than vasculature, retinal nerve fiber layer, and foveal pit reflex. The hyper-reflective features in the AOSLO images were grouped based on size, location, and subjective texture. Clinical imaging, including optical coherence tomography (OCT), scanning laser ophthalmoscopy, and fundus photography was analyzed for comparison.

Results: Seven categories of hyper-reflective inner retinal structures were identified, namely punctate reflectivity, nummular (disc-shaped) reflectivity, granular membrane, waxy membrane, vessel-associated membrane, microcysts, and striate reflectivity. Punctate and nummular reflectivity also was found commonly in normal volunteers, but the features in the remaining five categories were found only in subjects with retinal or neurologic disease. Some of the features were found to change substantially between follow up imaging months apart.

Conclusions: Confocal reflectance AOSLO imaging revealed a diverse spectrum of normal and pathologic hyper-reflective inner and epiretinal features, some of which were previously unreported. Notably, these features were not disease-specific, suggesting that they might correspond to common mechanisms of degeneration or repair in pathologic states. Although prospective studies with larger and better characterized populations, along with imaging of more extensive retinal areas are needed, the hyper-reflective structures reported here could be used as disease biomarkers, provided their specificity is studied further.

Keywords: adaptive optics; inner retina; neuro-ophthalmology; ophthalmoscopy; retinal disease.

Figure 1

Representative images of the first four features: punctate reflectivity (A1–4), nummular reflectivity (B1–4), granular membrane (C1–4), waxy reflectivity (D1–4). Diseases in each group include, rubella retinopathy (A1), achromatopsia (A2), optic disk pit (A3), normal (A4), normal (B1), glaucoma (B2), normal (B3), multiple sclerosis (B4), diabetic retinopathy (C1), Parkinson's (C2), branch retinal vein occlusion (C3), optic atrophy (C4), cone dystrophy (D1), central serous retinopathy (D2), birdshot choroidoretinopathy (D3), age-related macular degeneration (AMD, [D4]). (B4) is a contiguous vertical montage split into two halves, top is on the left and bottom is on the right. The first column of each row is highlighted below in Figures 3, 5, 7, 9. Scale bars: 100 μm.

Figure 2

Representative images of the final three features: vessel-associated membrane (E1-4), microcysts (F1–4), striate reflectivity (G1–4). Diseases in each group include Leber's congenital amaurosis (E1), Stargardt's (E2), macular hole (E3), diabetic retinopathy (E4), macular telangiectasia (F1), glaucoma (F2), optic neuritis (F3), optic atrophy (focused on photoreceptors) (F4), Best's disease (G1), choroideremia (G2), commotio retinae (G3), unknown retinopathy (G4). The first column of each row is highlighted below in Figures 12, 13, 15. Scale bars: 100 μm.

Figure 3

Multimodal imaging of punctate retinopathy example (A1), rubella retinopathy JC_0830. (A) Fundus photo with outline of AOSLO imaging. (B) The AOSLO image showing many small punctate reflective structures approximately 5 μm across. These structures appear to coalesce at the vessel creating what appears to be a membrane in (C, D). Scale bar: 100 μm. Of interest here are the small structures themselves, as they are found in a variety of other diseases and normal (Figs. 1A2–A4). (C) Fundus image from scanning laser ophthalmoscope (SLO) of the Cirrus OCT. Scale bar: 200 μm. (D) En face OCT segmented at the level of the vasculature (horizontal arrows). Square shows AOSLO imaging region of interest, horizontal line indicates location of OCT B-scan in (E). Scale bar: 200 μm. (E) OCT B-scan showing hyper-reflectivity surrounding a retinal vessel. Scale bar: 100 μm.

Figure 4

Follow-up of punctate hyper-reflectivity, an enlarged portion of the lesion shown in Figure 3. In the 18 months between the first imaging session (A), and the second imaging session there are few, if any, punctate structures that have not changed position. The lesion appears to be enlarging along the vessels, and contracting, such that the vessel no longer follows its original course, as illustrated by the dashed lines in (B). Scale bar: 50 μm.

Figure 5

Multimodal imaging of nummular reflectivity example (B1), normal subject JC_0007. (A) Fundus photo with outline of AOSLO imaging. (B) The AOSLO image showing many reflective structures 10 to 30 μm across glistening on the surface of the NFL. Scale bar: 100 μm. In this image the largest dot is approximately 24 μm in diameter (arrow), and the smallest is only 13 μm (arrowhead). (C) The LSO fundus image does not resolve the dots. Scale bar: 200 μm. (D) En face OCT segmented at the level of the ILM (horizontal arrows) also cannot resolve dots. Scale bar: 200 μm. (E) The OCT B-scan does not clearly resolve the small dots. Scale bar: 100 μm.

Figure 6

AOSLO follow-up imaging of nummular reflectivity in (B1), normal subject JC_0007. (A) Initial imaging. (B) A 2.5 month imaging reveals that nearly all of the dots have not moved or changed in appearance. Arrows depict a dot that was identified initially but not in follow-up. Scale bar: 100 μm.

Figure 7

Multimodal imaging of granular membrane example (C1), diabetic retinopathy RS_1007. (A) Fundus photo with outline of AOSLO imaging does not reveal any membrane. (B) The AOSLO image shows a meshwork of small reflective granules, although highly reflective, the retinal vessel is clearly visible underneath. Scale bar: 100 μm. (C) The SLO fundus image shows a glistening membrane (arrow). Scale bar: 200 μm. (D) En face OCT segmented at the level of the ILM (horizontal arrows) also resolves the thin membrane (arrow). Scale bar: 200 μm. (E) The OCT B-scan shows a small glistening reflection on the surface of the NFL (arrow). Scale bar: 100 μm.

Figure 8

Illustration of how the specular reflectivity of waxy membranes can lead to dramatic image intensity changes relative to the surrounding structures with eye motion. (A, B) are single frames taken from the same image sequence in subject KS_0625 with cone rod dystrophy (Fig. 1D4). The relative brightness of the membrane (lower right) changes substantially due to an 80-μm retinal movement. An example from subject DLAB_0029 with glaucoma shows a similar phenomenon caused by a 52-μm retinal movement (C, D). Each panel is 100 μm across.

Figure 9

Multimodal imaging of waxy membrane example (D1), cone dystrophy KS_1154. (A) Fundus photo shows a yellowish membrane in the region within the region of AOSLO imaging. (B) The AOSLO image shows a clumpy, highly reflective membrane on the surface of the retina, obscuring the NFL underneath. Scale bar: 100 μm. (C) A SLO fundus image shows a highly reflective membrane covering a large portion of the superior portion of the image field. Scale bar: 200 μm. (D) En face OCT segmented at the level of the ILM (horizontal arrows) also resolves the extensive hyper-reflective membrane. Scale bar: 200 μm. (E) The OCT B-scan shows a thick hyper-reflective membrane on the surface of the NFL. Scale bar: 100 μm.

Figure 10

Example of waxy membrane with notable contraction in normal subject JC_10146 OD. (A) En face OCT segmented at the level of the ILM shows a hyper-reflective membrane and contraction lines superonasal to the foveal pit. (B) The OCT B-scan through the membrane shows a hyper-reflective layer at the ILM spanning over the NFL. Arrowheads indicate extent of AOSLO imaging shown in (D). (C) An OCT B-scan outside of the membrane shows an abnormal peaked appearance to the NFL. (D) The AOSLO imaging on and below the membrane shows the clumped waxy appearance of the membrane, as well as the linear radial shadowing in the NFL caused by the contraction of the membrane. Scale bars: 200 μm.

Figure 11

Two month follow up of a waxy membrane in a glaucoma patient (DLAB_0029). Despite the absence of contractile membrane (see Fig. 10), the membrane has changed in appearance significantly over a short period. Scale bar: 100 μm.

Figure 12

Multimodal imaging of vessel-associated membrane example (E1), Leber's congenital amaurosis JC_0579. (A) Fundus photo appears normal. (B) An AOSLO image shows capillary loops entirely coated with a hyper-reflective membrane. Scale bar: 100 μm. (C) The SLO fundus image shows no obvious pathologic changes. Scale bar: 200 μm. (D) En face OCT segmented at the level of the GCL (horizontal arrows) shows hyper-reflective and disorganized vasculature. Scale bar: 200 μm. (E) The OCT B-scan shows many hyper-reflective spots (arrows), corresponding to hyper-reflective vessels. Scale bar: 100 μm.

Figure 13

Multimodal imaging of microcysts example (F1), macular telangiectasia subject JC_10075. (A) Fundus photo does not resolve microcysts. (B) The AOSLO image shows a scattered distribution of very small (arrowheads) to very large microcysts (arrows). Scale bar: 100 μm. The borders of the cysts appear darker than the surrounding structure, and nearly all have a bright reflex on their apex. (C) The SLO fundus image shows disordered reflectivity, but no microcysts. Scale bar: 200 μm. (D) En face OCT segmented at the level of the INL (horizontal arrows) resolves only the largest microcysts (arrows). Scale bar: 200 μm. (E) The OCT B-scan shows the same two very large microcysts (arrows) seen in AOSLO imaging. Scale bar: 100 μm.

Figure 14

A 13-month follow-up of microcysts found in subject KS_1100 with dominant optic atrophy, showing no obvious changes in numbers or appearance. Scale bar: 50 μm.

Figure 15

Multimodal imaging of striate reflectivity example (G1), Best's disease KS_0601. (A) Fundus photo shows the large vitelliform lesion just temporal to the macula. (B) The AOSLO image shows the NFL coursing horizontally, while a striped reflective structure runs from vertically. Scale bar: 100 μm. (C) The SLO fundus image also resolves the vitelliform lesion, but not the vertically oriented fibers. Scale bar: 200 μm. (D) En face OCT segmented along the contour of the Henle fiber layer (horizontal arrows) shows a rim of bright Henle fiber reflectivity surrounding the vitelliform lesion. Scale bar: 200 μm. (E) The OCT B-scan shows regions of increased Henle fiber reflection on either side of the vitelliform lesion (arrows). Scale bar: 100 μm.