AOIP@ARVO 2014

May 3, 2014, Orlando, Florida - ISIE Imaging Conference

May 4-8, 2014, Orlando, Florida - ARVO Annual Meeting


The AOIP had a strong 2014 presence at the ISIE and ARVO meetings, two of the premier gatherings of the vision research community.  Below is a list of presentations by AOIP members and our collaborators.

Click here for a PDF of our schedule.


  Paper (Program# - Room#)


  Poster (Program# - Poster#)


SESSION TIME SATURDAY 5/3 SUNDAY 5/4 MONDAY 5/5 TUESDAY 5/6 WEDNESDAY 5/7 THURSDAY 5/8
8:30 - 10:15am    
10:30am - 12:45pm


1:30 - 5:30pm
 
 
 
 

 

ARVO Abstracts

See our publications page for links to the IOVS online abstracts.

Sajdak, B., Cooper, R.F., Bazan, K., Higgins, B., Scoles, D.H., Wilk, M., Dubra, A., Carroll, J. “Improving the processing and analysis workflow of adaptive optics retinal imaging”

Purpose:

While adaptive optics (AO) retinal imaging enables high-resolution, in vivo, visualization of retinal pathology, the time required to process and analyze images is prohibitive for widespread adoption of this technology. Here, we sought to expedite two of the more time-intensive aspects of the workflow: image montaging and selecting regions of interest (ROIs) for subsequent density analyses.

Methods:

Six subjects with normal vision and 3 subjects with albinism were imaged using an AO scanning light ophthalmoscope (AOSLO). Sets of 18-26 partially overlapping images were aligned manually in Adobe Photoshop (San Jose, CA) and also semi-automatically with i2k Retina (DualAlign LLC, Clifton Park, NY) montaging software. We selected 100 x 100µm ROIs both manually and with custom software at 9-12 parafoveal locations. Montaging and ROI selection methods were timed to evaluate efficiency of manual and semi-automated techniques. For each ROI, cone density was measured to evaluate the accuracy of the semi-automated workflow.

Results:

Using i2k Retina reduced montaging time by 7-53 minutes, or 25-79%. The semi-automated ROI selection software reduced processing time by between 18-63 minutes, or 76-90%. Therefore, the semi-automated workflow resulted in a combined reduction of 29-75 minutes, or 50-80%. Cone density measurements obtained from the semi-automated workflow were on average 2,563 cones/mm2 less than those obtained using the manual workflow (95% CI = -9,465 to 4,339 cones/mm2, or 11 to 23%).

Conclusions:

While the time-saving benefits of automated image processing methods are appealing, accuracy appears to suffer. A current limitation of i2k Retina is that montages need to be assembled in piecemeal; further time savings could be realized if no user oversight was required. The ROI selection software was used for comparison of montaging techniques, and greatly improved the speed of the workflow process. The semi-automated workflow greatly reduced the time required for analysis, however, the discrepancy in cone density measurements may be too large to enable immediate adoption in its' current form. This study demonstrates the potential for an automated workflow, and identifies important design improvements needed in future iterations of automated montaging approaches.
Severn, P., Dubis, A., Cooper, R.F., Carroll, J., Dubra, A., Ramsamy, G., Fitzke, F., Rubin, G., Egan, C., Michaelides, M “Clinical assessment and single cell imaging in unexplained vision loss”

Purpose:

Current clinical practice involves gross visual inspection and functional analysis to assess the causes of visual impairments and deficit. While these tests can define most clinical diagnoses, some remain unresolved. Here we applied cellular resolution imaging to examine retinal structure in a patient with an unexplained visual deficit.

Methods:

Standard clinical assessment including dense SPECTRALIS optical coherence tomography (OCT), extensive electrophysiology (full field ERGs, multifocal ERGs, diffuse flash stimulation, EOG, high contrast pattern reversal, photopic ON and OFF responses including S-cone ERG and colour contrast sensitivity analysis), psychophysics (Nidek MP1 photopic microperimetry and photopic 30-2 Visual Field on a modified Humphrey) were applied to assess visual symptoms. Blood tests were also used to assess potential autoimmune involvement. Cellular imaging was completed using custom built adaptive optics scanning light ophthalmoscopes (AOSLO) at the Medical College of Wisconsin and Moorfields Eye Hospital.

Results:

Extensive clinical evaluation revealed no abnormalities to explain the patient’s visual symptoms. Single test microperimetry showed normal or only subtle abnormalities that were inconsistent on retest. ERG results were normal for all tests. AOSLO imaging showed small areas reminiscent of small sub-clinical drusen (1). Examples of these structures are shown in Figure A&B.

Conclusions:

While a few abnormalities were shown with AOSLO, they did not completely explain the visual complaints. However, these structures were more reproducible and conclusive than standard clinical measures.
Rosen, R., Pinhas, A., Razeen, M., Shah, N., Gan, A., Bavier, R., Weitz, R., Carroll, J., Dubra, A., Chui, Y.T. “Quantitative insights into macular microvascularity using adaptive optics scanning light ophthalmoscopy fluorescein angiography”

Purpose:

Adaptive Optics Scanning Light Ophthalmoscopy (AOSLO), coupled with fluorescein angiography (FA) , is able to resolve dynamic cellular details of human retinal microvasculature in healthy and diseased eyes. Using quantitative image analysis, AOSLO provides a platform for characterizing retinal microvascular changes due to age, onset of disease or response to treatment. Here, we show how foveal avascular zone (FAZ), capillary density and lumen in diabetes (DR), central retinal vein occlusion (CRVO), and sickle cell retinopathy (SCR) are different from those in fellow eyes and/or normal eyes.

Methods:

During AOSLO FA imaging, simultaneous reflectance (790 nm) and fluorescence (488 nm) image sequences with 1.75° field of view were  stitched together to create microvascular maps of a 6° square region centered on the fovea.  AOSLO FA maps were skeletonized and divided into regions of interest (ROIs).  Vessel length (mm) and density (mm-1) were then calculated per ROI.  For FAZ attribute quantification, the FAZ was delineated manually creating an FAZ layer mask. Based on the masks, FAZ area (mm2), effective diameter (µm, diameter of a uniform circle derived from FAZ area), perimeter (µm) and tortuosity index (TI) were computed.

Results:

Comparison of FAZ parameters of area, diameter, and perimeter in normal and vasculopathic eyes showed large variations, with CRVO eyes having highest values followed by SCR, DR, and controls. Tortuosity Index (TI), however, was highest in DR eyes followed by SCR, CRVO  and controls.  CRVO fellow eyes all showed some capillary dropout near the FAZ with significant decrease in vessel length and density compared to control eyes. FAZ  mean capillary lumen diameter for early diabetic subjects was found to be 35% larger than that of the control subjects.

Conclusions:

Quantification of the microvascular geometry utilizing AOSLO in vivo microscopy shows significant potential for studying complex clinical questions involving retinal vascular diseases. This approach may help direct therapeutic interventions based upon fine numerical distinctions as opposed to clinical impressions derived from conventional clinical imaging modalities.
Razeen, M.M., Gan, A., Shah, N., Pinhas, A., Bavier, R., Liu, C.L., Cheang, E., Dubra, A., Chui, T.Y., Rosen, R.B. “In vivo microscopic spatial characterization of foveal microvasculature in healthy human subjects” Investigative Ophthalmology & Visual Science, 55:E-Abstract 253 (2014).

Purpose:

To characterize the topography of foveal vessel density in healthy human subjects using adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA).

Methods:

Ten eyes of 6 healthy subjects (mean age 23.2±1.5 years old) were imaged using AOSLO FA after oral fluorescein administration. Simultaneous reflectance (790nm) and fluorescence (488nm) image sequences were acquired using a 1.75° field of view. Registered averages were tiled together to create perfusion maps centered at the fovea (Fig.1A). Maps were then skeletonized and overlaid by a grid of equiangular octants and 8 consecutive annuli (A), 100µm thick, centered at the fovea (Fig. 1B).The grid was centered on the manually determined center of the darkest area of foveal avascular zone (FAZ) on reflectance AOSLO. Here, we report vessel density (mean±SD) by pooling the annuli within each octant into two regions; A1 (200-400Œºm) and A2 (400-800Œºm), chosen to minimize the effect of the FAZ (0-200Œºm) and because of a radial vessel density plateau at 400Œºm.

Results:

Mean vessel density at A1 was 24.3±6.4mm-1 while at A2 was 50.8±3.1mm-1 (n=10). Despite small inter-octant differences (<10%), statistically significant inter-octant variability was found (two way ANOVA; F(7)=3.65, p<0.005), with highest A2 vessel densities recorded at the superior and inferior octants and lowest at nasal and temporal octants (Fig. 2). Significant inter-subject variability was found (two way ANOVA; F(5)=7.85, p<0.00001), while vessel density differences between fellow eyes were found not to be significant (t(4)=-0.43, p=0.69).

Conclusions:

The vascular density normative data presented here and the successful systematic identification of all capillaries within the foveal region is a first step towards developing highly sensitive quantitative bio-markers of ocular and systemic disease that affects the retinal vasculature.
Esposti, S.D., Ba-Abbad R., Pack, A., Aboshiha, J., Sulai, Y.N., Dubra, A., Webster, A., Dubis, A.M., Carroll, J., Michaelides, M. “High-resolution imaging to probe retinal integrity in RPGR associated rod-cone dystrophy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 254 (2014).

Purpose:

In preparation for gene augmentation therapies there is need to characterize retinal structure and function in patients with X-linked Retinitis Pigmentosa (RP) due to mutations in the RPGR gene to determine viability of successful intervention, window of opportunity, and sensitive and reliable end-points.

Methods:

Three male patients (15, 20 and 43 years old) with molecularly confirmed RPGR-associated RP underwent clinical examination and retinal imaging including autofluorescence (AF) imaging, spectral domain optical coherence tomography (SDOCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). Retinal lamination was assessed using SDOCT and AOSLO was used to probe integrity of the photoreceptor mosaic and assess qualitative changes in reflectance.

Results:

Visual acuity in the youngest patients was 20/20 and in the eldest 20/60. This was consistent with the residual island of outer retinal architecture on SDOCT and relatively normal central macular AF. All subjects had a perifoveal ring of increased AF. Foveal cone topography on AOSLO was normal in the younger subjects, deteriorating rapidly away from the foveal centre. The older subject had a disrupted mosaic throughout the foveal region. At the boundaries of the intact photoreceptor mosaic RPE-appearing cells were observed. These corresponded well with the transition zone between normal macular AF and the ring of increased AF signal, and disappeared at the outermost edge of the high-density AF ring. Consistent with previous reports, the transition zones from normal to high AF and from intact photoreceptor mosaic to RPE cells correlated well with the drop off in outer retinal architecture observed on SDOCT.

Conclusions:

Despite X-linked RP being a rapidly progressive and early-onset form of RP, there is a window of opportunity for intervention that extends several decades. It will be important to undertake serial quantitative imaging to determine the rate of progression, which is likely highly variable, to identify potential participants who might be most likely to benefit and to characterise the most sensitive metrics to determine efficacy in a timely fashion. Our AOSLO imaging is currently limited to reflectance imaging of waveguiding photoreceptors, new split-detection AOSLO that can identify cone inner segments will be valuable to probe whether rescuable cones remain over regions of RPE-like cells at the edge of the photoreceptor mosaic.
Liyanage, S.E., Cooper, R.F., Ba-Abbad, R., Sulai, Y.N., Dubra, A., Dubis, A.M., Carroll, J., Michaelides, M. “Imaging photoreceptor structure in subjects with R9AP- and RGS9-associated retinal dysfunction (bradyopsia)” Investigative Ophthalmology & Visual Science, 55:E-Abstract 259 (2014).

Purpose:

Mutations in the genes RGS9 and R9AP cause an autosomal recessive cone dysfunction syndrome characterized by reduced central vision, mild photophobia, normal color vision, lack of nystagmus, and normal fundi. It has many similarities to the cone dysfunction syndrome Oligocone Trichromacy. Here we examined three subjects using a multi-modal imaging approach to characterize the degree of residual cone structure.

Methods:

Two sisters (23 years and 16 years old) from one family harboring a homozygous in-frame deletion (p.D32_Q34del) in R9AP, and a simplex male (62 years old) with a compound heterozygous mutation in RGS9 (p.R128X/p.W299R), underwent high-resolution quantitative retinal imaging. Spectral domain optical coherence tomography (SDOCT) was used to assess retinal lamination. Adaptive optics scanning light ophthalmoscopy (AOSLO) was performed to assess integrity of the photoreceptor mosaic and determine qualitative changes in reflectance.

Results:

SDOCT images from the two sisters showed normal macular lamination. The simplex observer had a focal area of foveal hyper-reflectivity in the right eye, with a normal appearance in the left eye. AOSLO imaging identified a small, focal hypo-reflective lesion (non-waveguiding cones) in the simplex subject's right eye; the photoreceptor mosaic in his left eye and in both eyes of the two sisters was normal. No other abnormal reflectance differences were observed in the subjects’ photoreceptor mosaics. It is plausible that given the unilateral findings in the simplex case that the focal foveal abnormality may not be related to Bradyopsia but may possibly be due to previous injury.

Conclusions:

Patients with either RGS9/R9AP-retinopathy (Bradyopsia) or Oligocone Trichromacy (OT) have very similar clinical phenotypes, characterized by stationary cone dysfunction, mild photophobia, normal color vision, and normal fundi. However, cellular imaging has shown very different cellular structures. Previous AOSLO imaging of OT revealed a sparse mosaic of cones remaining at the fovea, with no structure visible outside the central fovea; in direct contrast, the patients imaged in this study had a normal cone photoreceptor mosaic.
Ramsamy, G., Aboshiha, J., Rajendram, R., Sulai, Y.N., Carroll, J., Dubra, A., Michaelides, M., Dubis, A.M. “Persistent and reversible structural retinal disruption associated with selected outer retinopathies” Investigative Ophthalmology & Visual Science, 55:E-Abstract 262 (2014).

Purpose:

A wide range of acute onset outer retinopathies have been described, which following the acute phase, may either progress, or varying degrees of recovery may occur. Predictive factors to determine which subjects will recover or progress are not yet known. In this study we applied serial high-resolution imaging to three outer retinopathies to characterise detailed structural changes, which may potentially be predictive of progression or recovery.

Methods:

Subjects with Acute Macular Neuroretinopathy (AMN), Persistent Placoid Maculopathy (PPM) and Poppers Maculopathy were imaged longitudinally using spectral domain optical coherence tomography (SDOCT), fundus autofluorescence (AF) and confocal adaptive optics scanning light ophthalmoscopy (AOSLO).

Results:

Outer retinal disturbance was observed in all subjects at presentation. These changes included loss or marked disruption of outer retinal lamination on SDOCT, areas of reduced AF, and loss of wave-guiding photoreceptor structure on confocal AOSLO. The changes in outer retinal integrity over time were characterized with serial imaging. The outer retinopathy progressed in PPM, while the AMN and Poppers Maculopathy recovered a variable degree of outer retinal structure. In the PPM patient, the area of loss of retinal architecture increased and photoreceptor appearance on confocal AOSLO changed from a "swollen" appearance to loss of reflectivity, suggesting further deteriorating photoreceptor structure and function (associated with further loss in visual acuity). The AMN and Poppers patients both had a degree of recovery of wave-guiding photoreceptor cells on confocal AOSLO, improved retinal lamination on SDOCT, and decreased area of reduced AF, which was coupled with an increase in visual acuity.

Conclusions:

Current clinical tools such as AF imaging and SDOCT can be used to monitor disease in acute onset outer maculopathies. Cellular resolution analysis with confocal AOSLO allows more precision in identifying functional cones, but new split-detection imaging will likely prove even more helpful in identification of remaining cones that may be saved.
Ba-Abbad, R., Pack, A., Aboshiha, J., Sulai, Y.N., Dubra, A., Webster, A., Moore, A.T., Dubis, A., Carroll, J., Michaelides, M. “Outer retinal changes associated with the RPGR carrier phenotype: insights from high-resolution imaging” Investigative Ophthalmology & Visual Science, 55:E-Abstract 274 (2014).

Purpose:

Retinal structure and function in carriers of X-linked retinitis pigmentosa (XLRP) have been investigated previously, with an inconsistent correlation between the tapetal-like reflex seen on clinical examination and various imaging modalities. In this study we assessed the cone and rod photoreceptor mosaic in a cohort of unrelated carriers of RPGR-associated XLRP and examined retinal reflectivity using a multimodal imaging approach.

Methods:

Obligate carriers of XLRP were identified and underwent full ophthalmic examination including visual acuity, color fundus photography, fundus autofluorescence (AF) imaging, spectral domain optical coherence tomography (SDOCT), and adaptive optics scanning light ophthalmoscopy (AOSLO).

Results:

Five subjects with an age range of 28 to 62 years were examined. All had 20/20 visual acuity in both eyes, except for one patient who had unilateral amblyopia. Two patients described difficulties with night vision. In four patients a tapetal-like reflex (TLR) was clinically detectable bilaterally. The fifth subject had sparse bone-spicule retinal pigmentation. Fundus AF imaging ranged from normal to peri-macular areas of increased AF, with no definite co-localization with the TLR. SDOCT showed normal retinal lamination in all patients. AOSLO showed multiple small areas of variable rod loss and rod hyper-reflectivity, with normal appearing cones, in the 4 subjects with a TLR. The fifth subject with bone-spicule pigmentation had no evidence of rod loss or abnormality in rod reflectivity.

Conclusions:

We speculate that the rod hyper-reflectivity may underlie the TLR seen clinically. It is of note that no abnormality in rod reflectance was seen in the single obligate carrier without a TLR. It remains to be established via serial assessments over time whether the degree of rod loss and photoreceptor reflectance disturbance have prognostic implications.
Wilk, M., Higgins, B., Cooper, R., Scoles, D., Stepien, K., Summers, C.G., Dubra, A., Costakos, D., Carroll, J. “Contrasting foveal specialization in disorders associated with foveal hypoplasia” Investigative Ophthalmology & Visual Science, 55:E-Abstract 694 (2014).

Purpose:

While foveal specialization has been well characterized in albinism, less is known regarding foveal morphology in other disorders associated with foveal hypoplasia. Here we sought to quantify foveal specialization in patients with aniridia or a history of premature birth using spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO), and compare these findings to those of patients with albinism.

Methods:

Subjects with a diagnosis of albinism (n=5), aniridia (n=3), or a history of premature birth (n=3, birth was between 25 and 30 weeks’ gestation) were recruited for this study. Volumetric SD-OCT scans of the macula were acquired, and custom MATLAB software was used to derive estimates of foveal pit depth, diameter, and volume. Additionally, high-resolution linear SD-OCT scans were acquired and manually segmented to obtain measurements of relative foveal cone inner and outer segment (IS and OS, respectively) length. Images of the photoreceptor mosaic and foveal avascular zone (FAZ) were acquired using AOSLO. When possible, cone density was measured using a semi-automated cone counting program, and FAZ area and diameter were measured using semi-automated segmentation.

Results:

Despite having reduced FAZ areas, all 3 subjects with a history of premature birth displayed normal foveal pit metrics, normal foveal cone OS elongation, and normal cone packing. Consistent with previously reported results from 32 subjects with albinism and the additional 5 subjects reported here, the subjects with aniridia had variable OS lengthening. Clear evidence of cone packing was seen in one subject with aniridia, though due to the presence of severe nystagmus, it was not possible to quantify cone density in the remaining two subjects.

Conclusions:

Contrary to previous observations in patients with a history of premature birth, our subjects displayed normal foveal specialization. Further work contrasting foveal specialization across these disorders may be useful to better understanding normal foveal development. While previous work from our group has shown that it is possible to obtain high quality images in patients with nystagmus, AOSLO hardware improvements and some form of eye tracking are needed to enable imaging of patients with severe nystagmus, such as that seen in aniridia.
Dubra, A. “Adaptive optics scanning light ophthalmoscopy: thinking outside the pinhole” Investigative Ophthalmology & Visual Science (2014).

Invited Speaker Presentation:

The development of ophthalmic adaptive optics (AO) to correct for the monochromatic aberrations of the eye gave rise to a new generation of high-resolution ophthalmoscopes. Among these, scanning ophthalmoscopes have always been attractive due to their axial sectioning and potentially superior transverse resolution. Assuming perfect AO correction, both these capabilities are determined by the size of the confocal aperture in front of the light detector. When using linear imaging modalities, the smaller the aperture (down to the size of an Airy disk), the higher the contrast of the features in focus due to the removal of out-of-focus light. Using apertures smaller than the Airy disk can provide an additional 15 to 20% lateral resolution improvement over Abbe’s diffraction limit. Therefore, the desire to improve signal-to-noise ratio and resolution led to the use of the smaller confocal apertures that provide sufficient signal, irrespective of whether using reflectance, single-photon fluorescence, time- or spectral-domain optical coherence tomography.
 
In AO scanning ophthalmoscopy, the information in the light outside the confocal aperture has been largely ignored, until recently. It was in fact only in 2012, that Chui et al. (Biomed. Opt. Express 3:10, 2012) demonstrated a de-centered circular confocal aperture in an AO scanning light ophthalmoscope (SLO), in a similar manner to the annular mask demonstrated in 1987 by Webb et al. (Appl. Optics 26:8, 1987) in an SLO. These imaging modalities, termed indirect illumination, Tyndall view, dark-field or retro-illumination, enhance the view of the transparent structures of the retina.
 
In this work we will review AOSLO detection configurations for visualizing the retinal pigment epithelial cell mosaic, cone photoreceptor inner segments and the retinal vasculature and its perfusion. This will be followed by a discussion of optimal detection schemes for the retinal structure of interest, by enhancing or attenuating the symmetric and anti-symmetric signals in the non-confocal region of the image plane. These include circular, annular, filament and knife-edge masks with a single light detector, as well as a non-confocal split detector detection scheme.
Flatter, J., Scoles, D., Cooper, R., Sulai, Y., Goldberg, M., Wirostko, W., Stepien, K., Connor, T., Dubra, A., Carroll, J. “Changes in outer retinal structure following closed globe blunt ocular trauma” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1090 (2014).

Purpose:

Imaging with adaptive optics scanning light ophthalmoscopy (AOSLO) has shown persistent photoreceptor mosaic disruption following ocular trauma, though only a single time point was examined. Here, we assess changes in photoreceptor structure over time following closed globe blunt ocular trauma and employ a novel AOSLO imaging modality to better delineate the extent of photoreceptor disruption in these patients.

Methods:

Two subjects with histories of visual complaints following ocular trauma were initially imaged between 4 and 6 months post trauma. To assess changes in outer retinal structure, imaging was repeated 4 to 15 months later. SD-OCT was used to acquire high-density volume scans through the fovea for assessment of retinal architecture. Confocal and split-detector AOSLO were used to visualize the waveguided and non-waveguided photoreceptor signals, respectively.

Results:

In one subject the area of the ellipsoid zone (EZ) disruption decreased between 6 and 21 months post trauma from 0.070 to 0.017mm2 on SD-OCT and from 0.075 to 0.029mm2 on confocal AOSLO. Confocal AOSLO imaging on a second subject revealed diffuse photoreceptor disruption that remained grossly unchanged between 4 and 8 months post trauma. In both subjects, visualization of the inner segments by split-detector AOSLO allowed for more definitive assessment of residual cone structure. In one subject, confocal AOSLO overestimated the focal lesion size by over 300% with respect to split-detector AOSLO (Figure). Split-detector imaging also revealed the presence of enlarged cones bordering the focal photoreceptor mosaic disruption (Figure), and microcysts in areas of parafoveal photoreceptor mosaic disruption visualized by confocal AOSLO.

Conclusions:

Assessment of the degree of residual cone structure following trauma is challenging when relying solely on confocal AOSLO imaging and/or en face SD-OCT. Split-detector imaging was able to disambiguate reflective signals derived from remaining cones from those originating from other retinal structures, and this technique may be useful as a prognostic indicator of expected recovery in these patients.
Langlo, C., Scoles, D., Fishman, G., Gamm, D., Struck, M., Chiang, J., Dubra, A., Carroll, J. “Residual cone structure in achromatopsia: implications for gene therapy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1101 (2014).

Purpose:

Achromatopsia (ACHM) is associated with absent or severely reduced cone function. Central to the success of emerging gene-replacement therapies is identifying patients with residual cone structure despite functional deficits. Adaptive optics (AO) imaging studies have shown that most residual cones have reduced or absent reflectivity, interfering with quantification of cone populations. Here we demonstrate a novel AO imaging method to visualize cones regardless of their waveguided signal.

Methods:

Twenty-three subjects with a clinical diagnosis of ACHM, 13 with confirmed genetic mutations, were imaged using confocal and split-detection AOSLO. Split-detection AOSLO enables visualizing structures using multiply scattered light. These images were captured concurrently, in exact spatial registration with one another. Cones with visible confocal signal were matched to locations of cone inner segments (IS) in split detection images. In regions of mismatch between the two images, cone IS diameters were measured in the split detection images.

Results:

All subjects showed regions of diminished or absent reflectivity as previously reported. Structures seen in three subjects using the split detector method were found to have a mean diameter of 6.0, 6.3, 6.7 and 7.0μm at 0.5, 5, 10 and 15° from the foveal center. The structures nearest the fovea were about twice the size of histologic values of cone IS diameter and change with increasing eccentricity was smaller than in histology. In images acquired with split-detection the location of many IS correspond to regions with no confocal signal (see Figure). IS structure was preserved throughout the retina in these three subjects, including a contiguous mosaic in the fovea. The split detector findings indicate that the regions of diminished reflectivity in the retinas of the 20 other subjects are likely to correspond to locations of cone IS.

Conclusions:

The split-detector AOSLO method allows for observation of a robust population of non-reflecting cones in subjects with ACHM. This ability is important for screening efforts, as emerging gene therapy trials will benefit from objective parameters defining the therapeutic potential of prospective participants.
Warren, C., Scoles, D., Dubis, A., Aboshiha, J., Webster, A., Michaelides, M., Han, D., Carroll, J., Dubra, A. “Imaging cone structure in autosomal dominant cone rod dystrophy caused by GUCY2D mutations” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1102 (2014).

Purpose:

Mutations in the GUCY2D gene are known to cause autosomal dominant cone rod dystrophy. Here we examined four subjects with GUCY2D gene mutations using a multi-modal imaging approach to characterize residual cone structure.

Methods:

Three family members spanning three generations and a single unrelated subject, who were all found to harbor the Arg838His substitution in GUCY2D and diagnosed with autosomal dominant cone rod dystrophy, were recruited for imaging. Spectral domain optical coherence tomography (SD-OCT) was used to assess outer retinal lamination. Adaptive optics scanning light ophthalmoscopy (AOSLO) was performed in three of the four subjects using confocal detection, while two of the subjects were also imaged using AOSLO split-detection.

Results:

SD-OCT findings were variable across subjects and included macular atrophy, the presence of a large hyporeflective zone, and subtle mottling of the ellipsoid zone. Confocal AOSLO revealed altered reflectivity of the perifoveal cone mosaic, showing sporadic dark cones throughout the perifovea, which were aided in visualization by the presence of neighboring rod photoreceptors. Confocal AOSLO near the fovea showed irregular reflective structure, precluding analysis of residual cone structure (see Figure). However, using AOSLO split-detection, we were able to clearly visualize cone inner segments. At 0.65° from the fovea, cone density was significantly reduced from normal (approximately 17,000 cones/mm2 in the two subjects compared to 72,500 cones/mm2 expected for normals), while residual cone inner segments were found to be enlarged.

Conclusions:

Interpretation of confocal AOSLO images of degenerative retinal disease can be challenging. AOSLO split-detection allows direct quantification of residual inner segment cone structure and is complementary to the confocal signal from the cone outer segments. These techniques should prove to be a powerful clinical tool to aid in the examination of cone rod dystrophies and other retinal disorders.
Smith, E.S., Chen, C., Chui, T.Y., Tsang, S.H., Carroll, J., Dubra, A., Hood, D.C., Rosen, R.B., Greenstein, V.C. “Comparison of adaptive optics scanning light ophthalmoscopic images to structure and function within and on the borders of hyperautofluorescent rings in retinitis pigmentosa” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1413 (2014).

Purpose:

To compare images obtained with adaptive optics scanning light ophthalmoscopes (AOSLO) to measures of visual function and retinal structure within the hyperautofluorescent ring where they are relatively preserved, and on the ring border in patients with retinitis pigmentosa (RP).

Methods:

A Canon prototype, AOSLO system 1 was previously used to obtain images from 15 eyes of 15 RP patients with visual acuities 20/20-20/30, and hyperautofluorescent rings (ARVO 2012). To aid in the interpretation of the photoreceptor mosaic, AO images were obtained in a subset of 5 patients along the horizontal meridian (fovea +/- 10 degrees) with Canon AOSLO prototype 2 and with a custom research AOSLO system.[1] In addition to AOSLO imaging, 10-2 visual fields (Carl Zeiss Meditec Inc.) and cone and rod mediated visual sensitivities were measured (Haag-Streit AG). Fundus autofluorescence and spectral domain-optical coherence tomography (OCT) line scans through the fovea were also obtained (SPECTRALIS HRA+OCT Heidelberg Engineering GmbH). The thickness of the total receptor layer (R+: Bruch's membrane to the border between the inner nuclear layer and outer plexiform layer) was measured using a computer-aided manual segmentation technique; values were compared to those for 30 age-similar normals.[2]

Results:

Within the ring, the inner segment ellipsoid band (ISe) was preserved and cone mediated visual sensitivity ranged from 0 to -6dB of normal. The AOSLO images from both systems showed dark areas/patches where photoreceptors appeared to be absent, this included the foveal area imaged with the higher resolution system where OCT R+ layer thickness and cone sensitivities were normal for 3 patients. On and outside the ring border, the ISe was absent, the R+ layer was significantly decreased, and there was marked cone sensitivity loss (13-35dB); images from both systems showed regions with ambiguous cone structure. With the higher resolution system, their size and shape suggest the presence of RPE cells with patches of preserved photoreceptors.

Conclusions:

Retinal imaging with AOSLO systems provides additional information about RP that is not apparent on OCT or visual function measures. 1. Sulai, Dubra. Biomed Opt Express. 2012, 3:1647-61. 2. Hood et al. IOVS 2009, 50:2328-36.
Stepien, K., Scoles, D., Sulai, Y., Cooper, R., Higgins, B., Carroll, J., Dubra, A. “Variability in photoreceptor inner segment morphology in best vitelliform macular dystrophy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1590 (2014).

Purpose:

Best Vitelliform Macular Dystrophy (BVMD) is an autosomal dominant macular degeneration characterized by macular vitelliform lesions. Previous adaptive optics scanning light ophthalmoscope (AOSLO) imaging revealed patchy photoreceptor mosaic disruption with areas of no waveguiding photoreceptors where vitelliform lesions are/had been present. However, it was unclear if these variations in photoreceptor reflectivity were due to loss of structure, change in orientation, or the inability to waveguide light. Here we apply a new imaging technique, split-detector AOSLO, to further characterize photoreceptor morphology in BVMD.

Methods:

Three affected family members with BVMD and known heterozygous BEST1 gene mutation (p.Arg218Cys) underwent comprehensive ophthalmic exams and high resolution retinal imaging. Outer retinal structure was assessed using spectral domain optical coherence tomography (SD-OCT), and photoreceptor mosaic was imaged with confocal and split-detector AOSLO.

Results:

SD-OCT confirmed patchy areas of outer retinal structure loss with focal areas of subretinal fluid and debris in areas with or with previous vitelliform lesions. Split-detector AOSLO revealed photoreceptor structure in areas where no waveguiding cones were visualized by confocal AOSLO (Figure 1), allowing for a more precise assessment of photoreceptor structure. Inner segment morphology varied significantly, ranging from a near-normal to enlarged, anomalously shaped inner segments with some having a long, tapering process extending from a more circular head (Figure 1B - arrow).

Conclusions:

When compared to SD-OCT or even confocal AOSLO, split-detector AOSLO allows for a more precise assessment of photoreceptor structure, highlighting the significant variability in inner segment morphology within areas of vitelliform lesions in BVMD. This demonstrates the potential utility of split-detector AOSLO for assessment of photoreceptor structure alterations in retinal degenerative processes such as BVMD.
Zakka, F., Scoles, D., Langlo, C., Liu, B., Han, D., Stepien, K., Connor, T., Dubra, A., Carroll, J. “Disambiguation of photoreceptor structure in transition zones of retinal degenerative diseases” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1591 (2014).

Purpose:

Inherited retinal degenerations are commonly associated with transition zones between normal and pathologic regions. Analysis of retinal structure within these zones provides important information about disease mechanism at the cellular level. Confocal adaptive optics scanning light ophthalmoscopy (AOSLO) imaging shows numerous reflective structures in these regions which may be erroneously identified as cones. This study uses non-confocal AOSLO imaging techniques to expose the origin of the reflective structures in transition zones and provide a more definitive assessment of residual cone photoreceptor structure.

Methods:

Two subjects with Stargardt Disease (STGD), 1 subject with retinitis pigmentosa (RP), and 1 subject with Usher's syndrome were imaged using confocal, dark-field, and split-detector AOSLO (custom-built, 790nm light). Spectral domain ocular coherence tomography (SD-OCT) images were acquired and aligned to the AOSLO.

Results:

At the transition zones of the RP and Usher's subjects, confocal AOSLO shows scattered reflective structures that are difficult to define (Fig 1A). Using dark-field and split-detector modalities reveals an increasingly visible RPE mosaic and a decreasing photoreceptor density, respectively, through the transition zone (Fig 1B, C). The loss of photoreceptors correlates with the tapering of the ellipsoid zone seen on SD-OCT in the same region (Fig 1D). Confocal AOSLO of the transition zone in the two patients with STGD showed numerous reflective structures that resemble cone photoreceptors (Fig 2A). Split-detector AOSLO of this region revealed a majority of rods, scarce cones, possible large lipid-engorged RPE cells, and a significant area of debris (Fig 2B).

Conclusions:

Reflective retinal structures in transition zones of inherited retinal degenerations could be wrongly attributed to photoreceptors when assessed solely with confocal AOSLO. Reflective RPE cells or debris can be better differentiated from surviving photoreceptors when a multimodal AOSLO imaging approach is applied in patients with these degenerative diseases.
Saito, K., Nozato, K., Suzuki, K., Roorda, A., Dubra, A., Song, H., Hunter, J.J., Williams, D.R, Rossi, E.A. “Rods and cones imaged with a commercial adaptive optics scanning light ophthalmoscope (AOSLO) prototype” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1594 (2014).

Purpose:

Our goal is to develop a commercial adaptive optics scanning light ophthalmoscope (AOSLO) that is compact, easy to use, and has comparable resolution to existing research AOSLOs. We previously demonstrated a prototype commercial AOSLO capable of axial sectioning and high resolution imaging across a large dioptric range using dual liquid crystal on silicon spatial light modulators (LCOS-SLMs) for wavefront correction. Here we present modifications to our system to increase resolution and improve image quality.

Methods:

The AOSLO was improved by: 1) increasing the maximum pupil size at the eye to 6.7 mm, thus providing 3 µm theoretical lateral resolution at 840 nm, 2) using the same light source for both imaging and wavefront sensing, 3) optimally arranging the LCOS-SLMs to modulate the phase of each polarization component independently and minimize the undesired effects of diffracted light and 4) implementing real-time closed-loop wavefront correction. Real-time software tracking of pupil size and position ensured correction stability. The relatively compact optical system (28"x18") uses a focusing lens to achieve a high dioptric range (-10D to +5D). We imaged 9 healthy human eyes at 0°, 1.7° and 3.5° on both the modified system and an original system to compare performance. 2 subjects were also imaged at 7° in the modified system only. Cones were identified by an experienced grader masked to the system of origin using a semi-automated method and computed nearest neighbor distance (NND).

Results:

It was difficult to determine the precise eccentricity at which cones became visible, as we did not sample continuously from the foveal center outwards. However, cones were visible in the images obtained at 0° fixation in 7/9 subjects in the modified system; for these eyes the mean eccentricity beyond which cones were visible was 0.41° (range:0.17°-0.60°). The mean of the 100 cones with the lowest NND for these eyes was 0.45 arcmin. In the original system, cones were not visible until some eccentricity >0.83°. Rods could be seen in the modified system in some images at ~7°.

Conclusions:

This work demonstrates the suitability of LCOS-SLMs for wavefront correction in a commercial AOSLO device. We successfully imaged both rods and foveal cones in some subjects by substantially improving the performance of our AOSLO system, which is now approaching the resolution of the best research-grade AOSLOs.
Eells, J.T., Abroe, B., Schmitt, H.M., Gonzalez-Quevedo, A., Summerfelt, P., Dubis, A.M., Carroll, J., Gopalakrishnan, S. “NIR photobiomodulation does not alter retinal function or morphology in non-dystrophic Sprague Dawley rats” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1910 (2014).

Purpose:

Previous studies in our laboratory have shown that 670nm and 830nm photobiomodulation (PBM) protects against retinal dysfunction and photoreceptor cell death in rodent models of retinal injury and retinal degeneration. The purpose of this study was to test the hypothesis that NIR PBM would not alter retinal function or morphology in a non-dystrophic Sprague-Dawley rat.

Methods:

All studies were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. Sprague Dawley (Harlan Sprague-Dawley, Madison, WI) rats were treated once per day with either 670nm or 830nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (QDI, Barneveld WI) from postnatal day p10 to p25. Sham-treated rats were restrained for 180 seconds, but not exposed to 670nm or 830nm light. Retinal function and structure were assessed at p30 by measuring photoreceptor function with electroretinography (ERG) and retinal morphology using spectral domain optical coherence tomography (SD-OCT).

Results:

Photon irradiation with 670nm or 830 nm light did not alter ERG parameters in SD rats compared to sham-treated control animals. ERG a-wave amplitude and latency and ERG b-wave amplitude and latency were not altered by NIR PBM treatment. Retinal imaging studies using SD-OCT imaging revealed no differences in the structural integrity of the retina in NIR PBM treated rats compared to sham-treated control animals.

Conclusions:

Our findings demonstrate the safety of 670nm and 830nm photobiomodulation applied to the retina in Sprague-Dawley albino rats. They confirm other experimental and clinical studies demonstrating an absence of adverse effects of photobiomodulation. Further, they provide essential safety data for the continued development and clinical application of PBM for the treatment of retinal degenerative disease.
Martin, J., Kim, B., Joshi, R., Han, G., Dubra, A., Morgan, J.I. “Longitudinal adaptive optics imaging of active and resolved central serous retinopathy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1655 (2014).

Purpose:

Central Serous Retinopathy (CSR) is a type of exudative retinal detachment that usually resolves spontaneously. This study examined CSR patients over time using multiple high-resolution imaging modalities to understand cone mosaic remodeling during and after subretinal fluid (SRF) resolution.

Methods:

6 active and 2 resolved CSR patients were imaged at baseline, and 3 active CSR patients were followed longitudinally at 6 weeks, 3 months and then every 3 months (ranging from 3-12 months) using adaptive optics scanning light ophthalmoscopy (AOSLO), spectral domain optical coherence tomography (OCT), and microperimetry (MP). At each visit, images were captured in areas overlying and adjacent to SRF. The cone mosaic was imaged using an AOSLO from Canon, Inc (Tokyo, Japan) and a custom AOSLO. Serial images were aligned using TrakEM2 (Institute of Neuroinformatics, University of Zurich) and Adobe Photoshop.

Results:

Areas where cones were separated from the retinal pigment epithelium (RPE) showed remodeling of the photoreceptor mosaic that continued months after SRF resolution. MP sensitivity was reduced 6-16dB (mean 10.2dB, SD 3.8dB) in areas of SRF but improved after SRF resolution and often was normal despite visible cone loss on AOSLO. The ellipsoid band (EB) on OCT was disrupted in areas overlying SRF and returned following SRF resolution. There was a trend toward improvement of visual acuity associated with SRF resolution and return of the EB on OCT. Hyperreflective clusters were visualized in the outer retina adjacent to SRF of two patients (mean diameter 19.1µm, SD 6.4µm). These varied in location over time and their disappearance was temporally associated with SRF resolution.

Conclusions:

Local disruption of the cone mosaic was observed following SRF resolution. The cone mosaic geometry varied over time in affected areas of diseased eyes, potentially showing remodeling of the cone mosaic as it reattaches to the RPE. The hyperreflective clusters may represent phagocytic cells engulfing cone outer segments while patients have SRF. The movement of clusters suggests motile cells and their size is similar to that of activated macrophages. The return of MP sensitivity to normal levels despite patchy areas of cone loss on AOSLO may be due to the relatively large stimulus area (~0.5 degrees). Future studies will continue to examine photoreceptor structure and function in retinal diseases with SRF.
Sulai, Y., Scoles, D., Dubra, A. “Visualizing retinal vasculature using non-confocal adaptive optics scanning light ophthalmoscopy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1656 (2014).

Purpose:

To explore non-invasive imaging of the retinal vascular structure and perfusion by non-confocal detection in adaptive optics scanning light ophthalmoscopy (AOSLO).

Methods:

Five detection methods were tested by placing different spatial filters and/or light detectors in retinal conjugate planes: circular mask, annular mask, circular mask with filament, knife edge and split-detection. The dimensions and geometry of the detection apertures were varied and the effects of illumination pupil apodization, polarized detection and four different illumination wavelengths (500, 600, 680 and 790 nm) were also evaluated. A side by side comparison of all the detection schemes was performed at identical foci and retinal locations, using the signal-to-noise ratio (SNR) along capillary cross-sections as performance metrics, in both image sequence averages (structure maps) and standard deviations (perfusion maps).

Results:

Detection apertures that include areas outside the confocal signal (1 Airy disk diameter) facilitate the visualization of capillary walls. In areas where the vascular structure is overwhelmed by the strong confocal signal from the nerve fiber layer, detection methods which reject the confocal signal allow visualization of all capillary beds. Of the four non-confocal detection methods investigated, split-detection (see figure 1) is superior in terms of contrast and SNR for both structural and motion contrast imaging at all retinal locations. Apodization of the illumination pupil and linearly polarized detection decrease the SNR of the split-detector images. Raw images with visible illumination show substantially noisier backgrounds in both reflectance and motion contrast than those collected using infrared light, potentially due to lower signal. Registered image averages with comparable SNR however, show that image contrast is indeed reduced when using visible wavelengths. The perfusion maps created using motion contrast derived from asymmetric non-confocal detection schemes, such as knife-edge and split-detection, have some predictable and repeatable artifacts (doubling of vessels).

Conclusions:

Non-confocal AOSLO imaging can reveal the structure and perfusion of all retinal capillary beds non-invasively, including that serving the highly reflective nerve fiber layer.
Rosen, R.B., Gan, A., Razeen, M.M., Pinahas, A., Cheang, E., Liu, C.L., Rostomian, L., Weitz, R., Dubra, A., Chui, T.Y. “Monitoring retinal vasculopathic changes over time using in vivo offset pinhole adaptive optics scanning light ophthalmoscopy” Investigative Ophthalmology & Visual Science, 55:E-Abstract 1657 (2014).

Purpose:

Offset pinhole adaptive optics scanning light ophthalmoscopy (OP AOSLO) of the retinal microvasculature allows non-invasive imaging of the retinal microvascular wall, lumen, and blood flow, without the use of an exogenous contrast agent. In this study, we used OP AOSLO to detect and monitor subclinical microvascular change over time in various retinal vasculopathies.

Methods:

Longitudinal OP AOSLO imaging was performed on 6 patients: 2 with diabetic retinopathy (DR), 2 with central retinal vein occlusion (CRVO), 1 with branch retinal vein occlusion (BRVO) and 1 with hypertensive retinopathy (HR). OP AOSLO was implemented using a 790 nm light by displacing the confocal detection aperture laterally, according to the orientation of the retinal blood vessels. An additional 850nm light source was used as a wavefront-sensing beacon. Image sequences were acquired using a 1° or 1.5° field-of-view at a frame rate of 15 Hz. After sinusoidal distortion and eye motion were removed, registered averaged images were stitched together to create larger DF microvascular maps. Serial maps from the same eyes, taken 1 week to 5 months, were qualitatively assessed for vasculopathic change over time.

Results:

All patients showed vasculopathic features including microaneurysms, lumen clots, vessel looping and sprouting, non-perfused vessels (endothelial sleeves), tortuous vessels, and irregular lumen diameters. In one patient, we were able to detect microvascular changes as early as 1 month after the first imaging session. Two of the patients imaged showed no changes in microvascular structure (1 CRVO and 1 BRVO; Fig. 1A and B). In 4 patients, microvascular remodeling was observed, including microaneurysm regression (1 HR) and progression (1 HR and 1 CRVO), angiogenesis and capillary regression over time (2 DR) (F ig. 1C and D).

Conclusions:

OP AOSLO reveals subclinical microvascular changes cross-sectionally and longitudinally, offering a great opportunity for studying the natural progression of vasculopathy and the response to treatment.
Bavier, R.D., Razeen, M.M., Gan, A., Pinhas, A., Shah, N., Cheang, E., Liu, C.L., Chui, T.Y., Dubra, A., Rosen, R.B. “In vivo microscopic assessment of perfused foveal capillary lumen diameters in diabetic retinopathy versus healthy controls” Investigative Ophthalmology & Visual Science, 55:E-Abstract 2602 (2014).

Purpose:

To measure the lumen diameter of perfused capillaries surrounding the foveal avascular zone (FAZ) and assess the variability in diabetic retinopathy (DR) versus healthy control eyes.

Methods:

Offset Pinhole (OP) Adaptive Optics scanning light ophthalmoscopy(AOSLO) imaging was performed on 5 subjects with DR (mean age 47, 35-61 years old) and 4 healthy controls (mean age 29, 25-37 years old). OP AOSLO used a 790 nm light source and a 1.5° field-of-view. Whenever used, AOSLO FA was performed using simultaneous reflectance (790 nm) and fluorescence (488 nm) image sequences with a 1.75° field-of-view after consumption of oral fluorescein. After sinusoidal distortion and eye motion correction, respective registered averages were stitched together to create structural (OP AOSLO) and perfusion (AOSLO FA) maps. Structural and perfusion maps were compared to identify the perfused capillaries surrounding the FAZ (Fig. 1). To ensure equal distribution of measurements, structural maps were divided into equiangular octants and segments 15° apart centered at the fovea. Lumen diameter measurements on perfused capillaries in OP AOSLO structural maps were then performed manually on 3 segments per octant.

Results:

Results are presented as mean diameters± SD, and statistical significance was assessed using a two-tail t-test with a p-value <0.05. DR eyes had statistically significant higher perifoveal perfused capillary lumen diameters than those of control eyes (5.5 ± 2.2 μm vs. 4.2 ± 0.89 μm; t(204) = 5.2, p < 0.0001). Also, DR eyes exhibited greater lumen diameter variability with a larger coefficient of variation compared to that of control eyes (0.34 vs. 0.24).

Conclusions:

The ability to quantify changes to the FAZ capillary lumen diameter in subjects with DR may have clinical value for early detection of microvascular changes, enabling interventions prior to capillary decompensation.
Chui, T.Y., Gan, A., Razeen, M., Shah, N., Pinhas, A., Rostomian, L., Cheang, E., Liu, C.L., Dubra, A., Rosen, R.B. “Imaging retinal microaneurysms in diabetes using offset pinhole adaptive optics scanning light ophthalmoscopy: a quantitative and qualitative analysis” Investigative Ophthalmology & Visual Science, 55:E-Abstract 2606 (2014).

Purpose:

To investigate the association of retinal microaneurysm (MA) dimension, structural characteristics, and blood flow patterns in diabetes using an offset pinhole adaptive optics scanning light ophthalmoscope (AOSLO).

Methods:

MA imaging was performed on 7 diabetic patients (7 eyes, 3 males, age 50±10years) using an offset pinhole AOSLO with an imaging wavelength centered at 790nm. Image sequences were acquired using 1° or 1.5° field of view at a frame rate of 15Hz. After sinusoidal distortion and eye motion were removed, registered movies and averaged images were obtained for qualitative MA structural characteristic and blood flow pattern assessments. Quantitative measurements of MA total area (TA) and blood filled area (FA) were delineated manually on the averaged images using MATLAB (MathWorks, Natick, MA).

Results:

79 MAs were imaged in 7 eyes. 87% of the MAs showed visible vascular wall structure. Mean±SD (µm2) of MA total area and blood filled area was 2400±2370 and 1500±1700, respectively. The percent of FA/TA was 66±22% (range: 8% - 100%). 4 MA morphologies were observed (focal bulge, 16%; saccular, 57%; fusiform, 19%; irregular, 8%) (Fig. 1). There were significant differences between their mean measurements of TA (p=0.0004), FA (p=0.025), and FA/TA (p=0.038) (ANOVA test). 5 blood flow patterns were recorded (minimum disturbance, 37 %; disturbed laminar flow, 35%; whirlpool, 16%; throbbing, 6%; no flow, 6%) with significant difference between their mean measurements of TA (p<0.0001), FA (p<0.0001), and FA/TA (p=0.04) (ANOVA test). MAs with granular surface and background changes were associated with larger MA TA (p<0.001), larger FA (p<0.001), and smaller FA/TA (p<0.05) (unpaired t test). MAs with lumen clots and reflective wall structure were associated with larger MA TA (p<0.001) and larger FA (p<0.001) (unpaired t test).

Conclusions:

Offset pinhole AOSLO provides noninvasive and direct quantitative assessments of MA structural features and blood flow patterns in patients with diabetic retinopathy. This imaging technique allows for a better understanding of the dynamic interaction between vascular wall and in vivo blood flow.
Han, G., Sulai, Y.N., Maguire, A.M., Bennett, J., Dubra, A., Morgan, J.I. “Adaptive optics imaging of ABCA4 retinal degeneration” Investigative Ophthalmology & Visual Science, 55:E-Abstract 2616 (2014).

Purpose:

To characterize disease pathogenesis in Stargardt Retinal Dystrophy through high resolution confocal adaptive optics (AO) retinal imaging.

Methods:

10 Stargardt patients aged 11-64 with ABCA4 genetic mutations were imaged using AO scanning light ophthalmoscopy (AOSLO) (Canon, Inc and/or a custom system), spectral domain optical coherence tomography (OCT) (Heidelberg SPECTRALIS), autofluorescence (AF), fundus photography, wide-field P200C-AF (Optos, PLC) and fundus guided photopic microperimetry (Nidek MP1). Longitudinal AOSLO imaging was performed in two patients, at 10 and 24 months. Images from all modalities were co-registered in Photoshop. Cell density was measured in AOSLO images using a semi-automated Matlab script. Cell diameters were measured manually.

Results:

AF imaging revealed retinal pigment epithelial (RPE) atrophy in all patients including hypo-AF patches of RPE loss in central retina, with hyper-AF macular flecks (7/10 patients) and/or a hyper-AF ring surrounding the central atrophic region (4/10). Wide-field SLO showed the RPE atrophy and abnormal AF was confined to the central retina in all but 2 patients, one which exhibited hyper-AF mottling into the far periphery, and another with atrophy in the far temporal retina. Foveal sensitivity was reduced in all patients by at least 3dB, only 2 patients did not exhibit an absolute scotoma (sensitivity reduced by more than 2 log units) within 1mm of the fovea. 6 patients fixated foveally; 4 patients fixated with superior retina. In 4 patients who fixated foveally, AOSLO imaging revealed a contiguous foveal cone mosaic with substantially enlarged cones and reduced cone density (p<0.001). Qualitatively, cones observed eccentric to the fovea also appeared enlarged; however, quantitative cell density measurements many times produced results within or above normal cone density measures. Photoreceptor layers were present at these same locations on OCT.

Conclusions:

AOSLO imaging allows single cell analysis in ABCA4 retinal degeneration. Cone loss and enlargement can be observed at the fovea and occurs in parallel with changes in the RPE lipofuscin content. At eccentricities outside of the fovea, cell loss and enlargement may occur in both cone and rod photoreceptors - further study is needed to confirm this hypothesis. Future studies will further delineate pathogenesis in ABCA4 disease.
Cooper, R., Langlo, C., Scoles, D., Stepien, K., Connor, T., Dubra, A., Carroll, J. “Assessing photoreceptor reflectance changes in retinitis pigmentosa” Investigative Ophthalmology & Visual Science, 55:E-Abstract 2617 (2014).

Purpose:

It is well established that photoreceptor reflectance fluctuates over time when imaged with adaptive optics (AO) techniques, though the origin of these changes remains unclear. In order to elucidate the origin of these reflectance variations, we examine this variability in cone photoreceptors in subjects with retinitis pigmentosa (RP) compared to normal subjects.

Methods:

Four subjects with no known retinal pathology and 2 subjects with RP (1 simplex, 1 Usher Syndrome) were imaged using an AO scanning light ophthalmoscope (AOSLO). Image sequences of the parafoveal cone mosaic were obtained using a 790nm (12.1 μm coherence length) source every 5 minutes for 1 hour. Average images from each time point were aligned, and cell locations were determined. Temporal cone reflectance profiles were classified as flat, monotonic, oscillating or spiking, and compared using a chi-squared test for independence. Additionally, cone reflectance was compared using standard deviation and range over both time and spatially within each image. Images of cone inner segment structure were acquired using a split-detector AOSLO.

Results:

We analyzed 16,963 cones across all images. Cones in RP and normal subjects had significantly similar (p<0.0001) classifier distributions. The average standard deviation of spatial cone reflectance was greater in RP (32 and 31 AU) than in normal subjects (range: 18-26). The range of cone intensities was 193 and 214 AU in RP subjects, similar to normal subjects (range: 174-222). Temporal cone profiles had a standard deviation of 16 and 13 in RP and 12 AU on average in normal subjects (range: 10-14). Profiles had a range of 52 and 46 in RP subjects, and 40 AU on average in normal subjects (range: 35-48). Despite 159 cones in one RP subject lacking reflectivity in confocal AOSLO, intact inner segment structure was observed using split-detector AOSLO. Including these cells in the reflectance analysis resulted in an increase in spatial standard deviation (31.4 AU), and a decrease in temporal standard deviation (13.3 AU) and dynamic range (44 AU).

Conclusions:

The reflectance behavior of cones in subjects with RP appears different than in subjects without retinal pathology, suggesting that reflectance may be a useful biomarker in retinal diseases that affect the outer segment. Split-detection AOSLO imaging showed that failure to include non-reflective cells can lead to substantial errors in temporal or structural analyses.
Collery, R.F., Moehring, F., Cooper, R.F., Dubis, A.M., Carroll, J., Link, B.A. “Zebrafish as a model to study emmetropization, refractive error, and retinal substructure using spectral domain-optical coherence tomography” Investigative Ophthalmology & Visual Science, 55:E-Abstract 3035 (2014).

Purpose:

Spectral-domain optical coherence tomography (SD-OCT) accurately measures the anatomy and dimensions of the eye in vivo. Here, we characterize emmetropization of wild-type zebrafish, myopia onset in bugeye/lrp2 mutants, and visualize the highly ordered cone photoreceptor mosaic by SD-OCT. We combine high resolution visualization with an animal model amenable to genetic manipulation that can be used to study candidate genes for refractive error and other ocular diseases.

Methods:

Eye axial length, focal length and lens diameter were measured in wild-type and bugeye/lrp2 mutant zebrafish throughout their lifespan using a Bioptigen SD-OCT system. Cone photoreceptor mosaics were visualized using en face summed volume projection (SVP) images derived from the SD-OCT volume scans. Melanin synthesis was ablated in a subset of RPE cells using TALEN-mediated inactivation of tyrosinase.

Results:

We found that wild-type zebrafish became emmetropic by 1 month, while bugeye/lrp2 mutants were myopic, and worsened as they aged. Wild-type fish maintained emmetropia, and our data show that their lenses grow to balance the focusing power required as eye size increases. By generating SVP images at different retinal depths, we visualized the UV and S cone submosaics. Density measurements of these submosaics agreed with published values from histology. SVP images focused on the RPE layer showed regional melanin inhibition provided by the TALEN technique, with improved discrimination of the cone-RPE interface and underlying choroid and sclera in B-scans of 'windows' of non-pigmented RPE.

Conclusions:

As the zebrafish eye uses only lens refraction and axial length to control emmetropia, we can assay the effects of genes associated with myopia specifically on axial length modulation, the largest single contributor to refractive error. Changes in retinal morphology can be assessed during induction of blinding disorders by SD-OCT, and changes in cone density or patterning can be used to assess photoreceptor damage in visual disorders.
Pinhas, A., Gan, A., Razeen, M., Shah, N., Cheang, E., Liu, C.L., Dubra, A., Chui, T.Y., Rosen, R.B. “Fellow eye in retinal vein occlusion: in vivo microscopic analysis of foveal microvasculature” Investigative Ophthalmology & Visual Science, 55:E-Abstract 3825 (2014).

Purpose:

The prevalence of retinal vein occlusion (RVO) is estimated to be 0.5%, yet its pathogenesis is not completely understood. A number of systemic risk factors have been identified, and RVO may be a terminal thrombotic episode as a consequence of chronic pathological alterations of the vascular wall. In this study, we use adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA) to map and quantify the foveal microvasculature in affected and fellow eyes.

Methods:

For 2 central RVO (CRVO) fellow eyes (aged 55 and 28 years old), a superior hemicentral RVO (HRVO) affected eye, and its fellow eye (aged 39 years old), simultaneous reflectance (790nm) and fluorescence (488 nm) image sequences were acquired after fluorescein ingestion using a 1.75° field-of-view to map a 6° square area centered at the fovea. Respective registered averaged images were stitched together to create larger structural and perfusion maps, and compared against one another to identify capillary dropout. For quantitative analysis, perfusion maps were skeletonized and divided into equiangular octants within a 200-800-Œºm radial annulus centered at the fovea. Data from 0-200Œºm radially were not used to minimize effect of the foveal avascular zone. Total vessel length and average vessel density were then calculated per annulus for fellow eyes.

Results:

All 4 eyes showed capillary dropout (Fig. 1). Quantitative results are presented as mean ±SD. Statistical significance was assessed in comparison to previously acquired data on healthy control and CRVO affected eyes using a two-tailed t-test with a p-value <0.05. Compared to controls, fellow eyes showed a significant decrease in both total vessel length (p=0.0014) and average vessel density (p=0.0018) (Fig. 2). Furthermore, average vessel density of unaffected inferior octants of the superior HRVO eye resembled that of fellow eyes. Fellow eye measurements were greater than those of CRVO affected eyes, but this difference was not significant.

Conclusions:

Fellow eye changes may be reflective of chronic pathological processes occurring systemically, and may serve as an early indicator of pathology and increased risk of future venous occlusion.
Gan, A., Shah, N., Razeen, M.M., Pinhas, A., Cheang, E., Weitz, R., Gentile, R., Dubra, A., Chui, T.Y., Rosen, R.B. “In vivo microscopy using adaptive optics scanning light ophthalmoscope fluorescein angiography and analysis of the foveal microvasculature in sickle cell retinopathy and comparison with SD-OCT” Investigative Ophthalmology & Visual Science, 55:E-Abstract 3871 (2014).

Purpose:

To compare the foveal microvascular density in sickle cell retinopathy (SCR) with healthy controls using adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA), and to compare with SD-OCT retinal thickness profiles.

Methods:

AOSLO FAs of 3 SCR subjects (average age: 36 years; range: 32-42) using oral fluorescein (20 mg/kg) were acquired and compared to previously obtained healthy control data. Simultaneous reflectance (790nm) and fluorescence (488nm) image sequences covering a 6° square area centered at the fovea were acquired using a 1.75° field of view. Images were stitched together to create larger microvascular perfusion maps (Fig. 1A). The maps were skeletonized (Fig.1B) and Foveal Vessel Density (FVD) (mm-1) was calculated for a concentric annulus centered at the fovea (A=200-800µm). Inner Foveal Vessel Density (iFVD, 200-400µm radius) and Outer Foveal Vessel Density (oFVD, 400-800µm radius) were also calculated. To compare VD with retinal thickness, 12 SD-OCT radial scans centered at the fovea were acquired for each eye and segmented to measure inner retinal thickness (IRT), outer retinal thickness (ORT) and total retinal thickness (TT) at 50-750µm from the foveal center with a 100 µm interval. OCT thickness profiles were averaged. Statistical significance was assessed using an unpaired t-test.

Results:

Mean FVD was less in the SCR eyes compared to controls (34.8 ± 3.0 versus 41.8 ± 1.39 (mm-1), p=0.022). When comparing the inner and outer annuli, there was a significant decrease in the ORT thickness on SD-OCT in the outer annuli in SCR eyes versus controls (p=0.043). Although mean iFVD and oFVD in SCR were less than controls, the differences were not significant. (Fig.2).

Conclusions:

The reduction in SCR VD compared to controls is indicative of microvascular abnormalities occurring in the foveal region. Our results indicate that VD analysis of AOSLO FAs may be a sensitive tool to detect subclinical changes to the foveal microvasculature, showing promise to gain more knowledge about SCR. Further studies with a larger sample size are needed to correlate VD with OCT thickness profiles.
Chen, M., Chui, T.Y., Alhadeff, P., Ritch, R., Rosen, R.B., Hood, D.C., Dubra, A. “Imaging retinal nerve fiber bundles in glaucoma patients with deep local visual field damage of the macular region” Investigative Ophthalmology & Visual Science, 55:E-Abstract 4778 (2014).

Purpose:

To better understand the structural changes from relatively healthy to severely affected regions as seen on visual fields (VF) in glaucoma patients using an adaptive optics scanning light ophthalmoscope (AO SLO) and frequency domain optical coherence tomography (fdOCT).

Methods:

Nine eyes of 9 glaucoma patients with arcuate defects within the macula region as seen on 10-2 VF were tested as well as 4 eyes of 4 controls. Using a prototype AO SLO system, the retinal nerve fiber (RNF) bundles in a 20° vertical by 1° horizontal region centered at the fovea were imaged. High-resolution images, 1° by 1°, were montaged. RNF layer (RNFL) thickness was obtained from fdOCT vertical line scans after segmentation [1,2]. Using the fdOCT images, the RNFL was divided into 3 regions based upon a comparison to the RNFL thickness of 54 controls: 1. within normal limits (WNL), 2. severely affected (SA: little or no RNFL), and 3. transition zone (TZ), the region between the WNL and SA regions. To compare corresponding regions, the scale of the AO SLO and fdOCT images were corrected based upon axial length.

Results:

The average 10-2 VF total deviation values for the 3 OCT defined regions were: better than -3 dB (WNL); ≤-3 dB, but ≥-15 dB (TZ); and worse than -15 dB (SA). The RNF bundles in the WNL region were highly reflective and densely packed on the AO images; they appeared similar to controls (Fig 1A). In the TZ, RNF bundles were clearly present, but were less densely packed and less reflective (Fig 1B). In the SA region, RNF bundles, if present, were sparse and of low contrast (Fig 2A). In 2 eyes with deep VF loss, no RNF bundles were present, but circular structures about 14-28 µm in dia. were present (Fig 2B) and in 2 eyes, reflective membrane structures previously reported [3] were seen (Fig 2C). In general, the transition zone marked on the fdOCT scans agreed with the AO SLO images.

Conclusions:

Both structural tests, as well as the 10-2 VF, support the existence of a transition zone, where the sensitivity is reduced, the RNFL is thinned, and the RNF bundles appear abnormal. This region should be followed for progression as well as for neuro-protective changes. 1. Raza et al. AO 2011; 2. Yang et al. Opt Exp 2010; 3. Scoles et al. ARVO 2012
Harvey, Z., Dubra., A. “Motion distortion correction in scanning ophthalmoscopy by iterative image registration” Investigative Ophthalmology & Visual Science, 55:E-Abstract 4816 (2014).

Purpose:

Correction of distortion due to eye motion in scanning ophthalmoscope images is critical for multiple applications, including: increasing signal-to-noise ratio and/or speckle reduction through averaging, creating perfusion maps using motion contrast techniques and registration for longitudinal studies. Here, we demonstrate the improvement of a registration algorithm by applying it iteratively.

Methods:

An image registration algorithm (Dubra & Harvey, Lect. Notes Comput. Sc. 6204, pp. 60-71) that estimates motion by comparing image strips against a reference frame using a normalized cross correlation (NCC), was modified to: perform multiple iterations, achieve sub-pixel accuracy through NCC interpolation, and account for line skew. The algorithm was tested on image sequences from a confocal adaptive optics (A O) scanning light ophthalmoscope (SLO). Average NCC values of 30 images and the average strip displacement after registration were used as performance metrics.

Results:

Results from subjects with normal fixation (achromatopsia and controls) show that most of the quantitative and qualitative improvements take place in the first three iterations (figures 1 and 2 respectively). Actual values vary substantially across image sequences, but NCC increases in the order of 1.5% for iterative registration alone were measured, with an additional 0.5% when incorporation sub-pixel NCC maximum estimation and less than 0.2% due to skew correction. Sources of image variability such as tear film evaporation, poor and/or slow AO correction and electronics noise prevented achieving NCC values of one.

Conclusions:

Skew correction appears negligible in subjects with normal fixation, and it is expected to provide its most benefit in slower scanning modalities such as optical coherence tomography or in subjects with nystagmus. Both iterative registration and sub-pixel motion estimation translate in measurable improvement of the selected metrics, with their combination providing the best performance.
Shah, N., Pinhas, A., Gan, A., Razeen, M.M., Cheang, E., Liu, C.L., Dubra, A., Chui, T.Y., Rosen, R.B. “In vivo microscopy of the foveal avascular zone in normal and vasculopathic eyes” Investigative Ophthalmology & Visual Science, 55:E-Abstract 4535 (2014).

Purpose:

To map and analyze the geometrical descriptors of the foveal avascular zone (FAZ) and the tortuosity of the capillaries defining the FAZ boundaries in healthy controls and diseased eyes using the Adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA).

Methods:

AOSLO FA imaging was performed on 12 eyes of healthy control subjects (age mean 25, range 21-35), 4 eyes of patients with diabetic retinopathy (DR) (age mean 56, range 49-66; all treatment naïve), 4 eyes of patients with sickle cell retinopathy (SCR) (age mean 40, range 32-51; 3 treatment naïve, 1 post LASER) and 4 eyes of patients with central retinal vein occlusion (CRVO) (age mean 46, range 27-64; 1 treatment naïve, 3 post anti-VEGF). Simultaneous reflectance (790 nm) and fluorescence (488 nm) image sequences were acquired using a 1.75° field of view after oral fluorescein. Individual registered averages were tiled to create perfusion maps of the entire FAZ and its surrounding micro-vasculature. Manual delineation of the FAZ was used to calculate FAZ area (mm2), effective diameter (µm, diameter of the smallest circle to fully encompass the FAZ), perimeter (µm) and tortuosity index (TI).

Results:

Results are summarized in Table 1. Area, effective diameter and perimeter showed variation among normal and diseased eyes, with the CRVO eyes having significantly higher values than controls. The highest TI was seen in eyes with DR. No difference in metrics could be made between treatment naïve and treated eyes.

Conclusions:

Results concur with previous studies, and confirm the differences in FAZ geometry among normal and vasculopathic eyes. Our CRVO data corresponds with previous studies indicating that CRVO causes chronic and extensive capillary dropout resulting in a higher FAZ area, effective diameter and perimeter. The data also demonstrate that TI of DR FAZs was larger than TI of normal, SCR and CRVO eyes. As LASER and anti VEGF treatment do not seem to have an effect on the metrics, the changes in metrics over time could thus more accurately help track the pathology. The in vivo quantification of FAZ and microvascular geometry shows potential for detection and monitoring progression of vascular diseases in the eye.
Carroll, J., Scoles, D.H., Langlo, C.S., Neitz, J., Penesi, M.E., Neitz, M., Dubra, A. “Imaging cone structure in patients with OPN1LW and OPN1MW mutations” Investigative Ophthalmology & Visual Science, 55:E-Abstract 4542 (2014).

Purpose:

Adaptive optics (AO) retinal imaging in inherited color vision defects has shown that while most patients have normal contiguous cone mosaics, some can have mosaics of reduced density and/or cones with altered reflectivity. We examined the integrity of the cone mosaic in patients with OPN1LW and OPN1MW mutations, using a new split-detector AO scanning light ophthalmoscope (AOSLO) that captures multiply scattered light, enabling visualization of structures that scatter rather than directly reflect light (e.g., cone inner segments).

Methods:

Eight subjects were imaged using AOSLO with either confocal and/or split-detection: A male with deuteranopia due to a combination of polymorphic amino acids (LIAVA) in the OPN1MW gene, 2 males with blue cone monochromacy (BCM) due to a C203R substitution in both the OPN1LW and OPN1MW genes, 2 female carriers of BCM from the same family, and 3 males with normal color vision.

Results:

The male with deuteranopia had numerous gaps in his cone mosaic with confocal AOSLO, cone density was ~30% below normal. There was no change in cone density at 0.75° since the measurement 2 years ago (45,600 versus 44,000 cones/mm2), consistent with previous data showing no changes over an 8-year period. Split-detector AOSLO revealed that these gaps contained residual cone inner segment structure. In one of the males with BCM, we observed presumed cone inner segment structure throughout the fovea using the split-detector method (16,500 cones/mm2). In the BCM males and female carriers, confocal AOSLO images revealed gaps in the mosaic, and split-detector AOLSO revealed remnant inner segment structure in these gaps.

Conclusions:

Diminished reflective signal in confocal AOSLO images is likely indicative of disrupted outer segment structure. However, as shown by the split-detector AOSLO imaging, this does not mean there is absence of the photoreceptor cell. Split-detector AOSLO imaging will be essential for accurate assessment of cone disruption associated with various OPN1LW and OPN1MW mutations.
Dubis, A.M., Cooper, R.F., Carroll, J., Dubra, A., Michaelides, M. “Quantifying photoreceptor reflectance: when density is not enough” Investigative Ophthalmology & Visual Science, 55:E-Abstract 5201 (2014).

Purpose:

Current analysis of photoreceptor mosaics from adaptive optics images involves quantifying photoreceptor mosaic integrity either with metrics such as cone spacing and density or qualitative terms such as hyper- or hypo- reflective cells. Development of a metric to describe reflectance is important to monitor the integrity of individual cells in response to treatment.

Methods:

Eight subjects with a range of retinal pathologies were imaged using a custom-build adaptive optics ophthalmoscope. Images were registered and then averaged. To quantify reflectance, the local maximum was identified for 200 cones and 600 rods per subject. Locations were selected between 5-8 deg temporally with sufficient cones and rods, and free of vessel shadows. The profile was fit to a 2D Difference of Gaussian, to objectively define its border. Reflectance was taken as the average of reflectance values from the full width at half maximum of the profile. The reflectivity values for rod and cone photoreceptors were normalized to the average intensity of the image.

Results:

Single cases of Poppers Maculopathy, Bradyopsia and an RPGR carrier had normal cone and rod densities, and had > 89% of their photoreceptors with a normalized average reflectance (NAR)>1. The AMN patient had a reduction in cone density, with 60% of cones having a NAR>1; while 92% of rods were>1. Two other RPGR carriers had regions of both normal and decreased photoreceptor density. Interestingly, only 48% of the NAR>1, while 100% of the rods were>1. This is likely due to the observed rod hyper-reflectivity. Two patients with achromatopsia had 0% NAR>1, while 96% of rods were>1.

Conclusions:

Quantification of relative reflectance holds promise as a biomarker of photoreceptor health/disease across different diseases. This is particularly valuable given that gene augmentation therapies will not result in new cells and therefore geometrical descriptors of the photoreceptor mosaic will not be adequate to monitor therapeutic success/progression. Long- term usefulness of this metric will be based on longitudinal repeatability in normal and diseased retina.
Michaelides, M., Zakka, F.R., Aboshiha, J., Sulai, Y.N., Connor, T.B., Han, D.P., Dubra, A., Carroll, J., Dubis, A.M. “High-resolution imaging in Stargardt Disease: preliminary observations in preparation for intervention” Investigative Ophthalmology & Visual Science, 55:E-Abstract 5016 (2014).

Purpose:

There is considerable well-documented inter-subject variability in Stargardt disease (STGD) in terms of age of onset, and both pattern and rate of degeneration. Because of this variability and the lack of robust natural history data, longitudinal deep phenotyping is needed to better characterise the disease process. Such studies will result in a better understanding of the cellular changes associated with each genotype, which is a prerequisite to planned therapies for STGD.

Methods:

Eleven patients (ages 7 to 62) with molecularly confirmed STGD underwent examination and retinal imaging, including autofluorescence (AF) imaging, spectral domain optical coherence tomography (SDOCT), and confocal adaptive optics scanning light ophthalmoscopy (AOSLO). Serial imaging was undertaken over a period ranging between 3 months and 1 year. Retinal lamination was assessed using SDOCT, and AOSLO was used to probe integrity of the photoreceptor mosaic and assess qualitative changes in reflectance.

Results:

Visual function (20/20 to 20/200) and structure were highly variable in the cohort, with no consistent age-dependence. At presentation, the oldest subject in the cohort with a foveal-sparing STGD phenotype had normal foveal architecture, both on SDOCT and AOSLO, with only subtle parafoveal inner segment ellipsoid layer mottling and minimal mosaic disruption. At one year, the parafoveal disruption had progressed, with the foveal region remaining intact. Four subjects had relatively intact foveal architecture, with a ring of parafoveal photoreceptor loss. The remaining subjects had a variable degree of foveal atrophy, and a variable residual degree of parafoveal photoreceptor structure on AOSLO. The youngest subject was followed at three-monthly intervals, with progressive loss of photoreceptors detected with AOSLO, and increased atrophy on SDOCT. Areas of increased AF correlated with atrophic zones on SDOCT and areas of photoreceptor loss on AOSLO.

Conclusions:

High-quality serial photoreceptor mosaic imaging in STGD was able to detect progression in children as young as 7 years of age. Further longitudinal testing in larger cohorts of molecularly proven patients is necessary to identify patients who may be most suitable for intervention. This will also establish the natural history, rates of progression, and identify the most sensitive and accurate measures of treatment effect.
Gopalakrishnan, S., Abroe, B., Schmitt, H.M., Gonzalez-Quevedo, A., Summerfelt, P., Dubis, A., Carroll, J., Eells, J.T. “Different treatment paradigms of 830 nm photobiomodulation are retinoprotective in a rodent model of retinitis pigmentosa” Investigative Ophthalmology & Visual Science, 55:E-Abstract 5760 (2014).

Purpose:

Previous studies in our laboratory have shown that 830 nm photobiomodulation (PBM) protects against retinal dysfunction and photoreceptor cell death in the P23H rat model of retinitis pigmentosa when administered early in the course of the disease. The purpose of the present study was to determine if 830 nm PBM would continue to protect against retinal dysfunction and photoreceptor cell death as the animals matured.

Methods:

All studies were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. P23H [Line 1] rats were treated once per day with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (QDI, Barneveld WI) from [1] postnatal day p10 to p25, [2] p10 to p40 or [3] p20 to p40. Sham-treated rats were restrained for 180 seconds, but not exposed to 830 nm light. Retinal structure and function was assessed at p30 or p45 by measuring photoreceptor function with electroretinography (ERG), and retinal morphology using spectral domain optical coherence tomography (SD-OCT).

Results:

830nm PBM preserved retinal function and retinal morphology in 830 nm light-treated animals in comparison to the sham-treated group in each treatment protocol. Scotopic full-field flash ERG responses over a range of flash intensities (from 10 to 25000 mcd.s/m2) were greater in 830 nm light-treated P23H rats compared to sham-treated P23H rats. SD-OCT imaging showed that 830 nm PBM also preserved the structural integrity of the retina in each treatment protocol.

Conclusions:

Our findings confirm that the retinoprotective effects of 830 PBM observed early in the course of retinal degeneration in the P23H rat persist as the animals mature. Based on our findings and on other studies documenting the neuroprotective actions of PBM in experimental and clinical studies, we propose that photobiomodulation is an innovative, non-invasive therapeutic approach for the treatment of retinal degenerative disease.
Scoles, D., Goldberg, M., Langlo, C., Sulai, Y., Stepien, K., Weinberg, D., Kim, J., Dubra, A., Carroll, J., Blodi, B. “Non-invasive evaluation of microscopic retinal pathology in macular telangiectasia type 2” Investigative Ophthalmology & Visual Science, 55:E-Abstract 5951 (2014).

Purpose:

To characterize photoreceptor, retinal pigment epithelium (RPE) and vascular changes in macular telangiectasia (MacTel) type 2.

Methods:

Seven subjects, average age 63 years, diagnosed with MacTel type 2 at various stages were imaged in one eye using confocal adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). In addition, split-detection AOSLO was used in four subjects to visualize the photoreceptor inner segments, vasculature, and inner retina, while dark-field AOSLO was used to image the RPE mosaic. Vascular perfusion maps were generated as the standard deviation of split-detection AOSLO image sequences. The area of ellipsoid zone (EZ) lesion was measured with en face SD-OCT.

Results:

SD-OCT revealed disruptions of the EZ in 6 of 7 eyes (average 0.074mm2), with corresponding dark-regions of reduced or complete loss of photoreceptor reflectivity in confocal AOSLO imaging. RPE cells were seen with dark-field AOSLO in the center of two of the EZ lesions, where the photoreceptors appear to have degenerated. Split-detection revealed swollen cone inner segments on the edges of these lesions in 3 of 4 imaged patients, as well as microcysts ranging in size from 2 to 100 µm in all subjects, which were not always visible in SD-OCT. Fibrotic-like structures on the inner retinal surface as well as sharply demarcated cavitations were present in all subjects. Vascular perfusion maps revealed abnormally enlarged and tortuous vessels in all subjects and microaneurysms in two out of four subjects over the central 3° around fixation.

Conclusions:

The combination of multiple AOSLO imaging techniques with SD-OCT is useful for non-invasive study of the micro-pathology in MacTel. Specifically, dark-field AOSLO disambiguates the reflectivity inside and surrounding EZ lesions identifying reflective structures as RPE cell granules, while split-detection AOSLO reveals photoreceptor swelling and microaneurysms, which could be useful as biomarkers of disease activity and progression.

 

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