PURPOSE. Adaptive optics scanning light ophthalmoscopy (AOSLO) paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of neuronal activity in retinal ganglion cells (RGCs) at single cell resolution in the living eye. However, the inner limiting membrane (ILM) restricts viral transduction to the fovea in humans and non-human primates, hindering both therapeutic intervention and physiological study of the retina. To address this issue, we explored peeling the ILM before intravitreal injection to expand calcium imaging beyond the fovea in the living primate eye. METHODS. Five eyes from three Macaca fascicularis (aged 3-10 years; 2 males, 1 female) that were immune suppressed with cyclosporine, underwent vitrectomy and ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8. RGC responses to visual flicker were evaluated using AOSLO calcium imaging 1 to 6 months after intravitreal injection. RESULTS. Calcium activity was observed in RGCs throughout the ILM peeled area in all eyes, representing a mean eight-fold increase in accessible recording area relative to a representative control eye. RGC responses in the ILM peeled and control eyes were comparable and showed no significant decrease over the 6 month period after the procedure. In addition, we demonstrated that activity can be recorded directly from the retinal nerve fiber layer. CONCLUSIONS. Peeling the ILM is a viable strategy to expand viral access to RGCs for gene therapy, and when paired with GCaMP imaging has the potential to advance visual neuroscience, preclinical evaluation of retinal function, detection of vision loss, and assessment of therapeutic interventions.

Viral expression of the calcium indicator GCaMP in primate RGCs has enabled optical readout of retinal function at a cellular scale in vivo. To date, functional recording has been limited to transduced RGCs close to the foveal pit. In this study we evaluate ILM peel as a strategy to expand the area of transduced RGCs and allow functional recording beyond the fovea in the living eye. 4 eyes of 3 immunosuppressed macaca fascicularis received a 9-12° ILM peel centered on the fovea, followed by intravitreal injection of GCaMP8s 4-8 weeks post-peel. A 660nm flickering visual stimulus drove RGC GCaMP responses which were recorded with fluorescence adaptive optics scanning laser ophthalmoscopy. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in the area of expression relative to a control eye with no peel. Functional responses were obtained from RGCs at max eccentricities of 11.7 o, 8.0 o, 9.7 o, and 13.7 o and could be classified as ON or OFF types up to the edge of the peel. Mean RGC responses in ILM peeled and control eyes of the same animal were comparable at 3.5 o and longitudinal tracking of individual RGCs showed stable responses up to 6 months post-peel. ILM peel substantially expands the region of primate retina accessible for in vivo GCaMP beyond the foveal ring of RGCs. This presents new opportunities for physiological study of the retina and pre-clinical testing of novel therapies in retinal degeneration models.

All retina-based vision restoration approaches rely on the assumption that photoreceptor loss does not preclude reactivation of the remaining retinal architecture. Whether extended periods of vision loss limit the efficacy of restorative therapies at the retinal level is unknown. We examined longterm changes in optogenetic responsivity of foveal retinal ganglion cells (RGCs) in non-human primates following localized photoreceptor ablation by high-intensity laser exposure. By performing fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) of RGCs expressing both the calcium indicator GCaMP6s and the optogenetic actuator ChrimsonR, it was possible to track optogenetic-mediated calcium responses in deafferented RGCs over time. Fluorescence fundus photography revealed a 40% reduction in ChrimsonR fluorescence from RGCs lacking photoreceptor input over the 3 weeks following photoreceptor ablation. Despite this, in vivo imaging revealed good cellular preservation of RGCs 3 months after the loss of photoreceptor input, and histology confirmed good structural preservation at 2 years. Optogenetic responses of RGCs in primate persisted for at least 1 year after the loss of photoreceptor input, with a sensitivity index similar to optogenetic responses recorded in intact retina. These results are promising for all potential therapeutic approaches to vision restoration that rely on preservation and reactivation of RGCs.

Optogenetic therapies for vision restoration aim to confer intrinsic light sensitivity to retinal ganglion cells when photoreceptors have degenerated and light sensitivity has been irreversibly lost. We combine adaptive optics ophthalmoscopy with calcium imaging to optically record optogenetically restored retinal ganglion cell activity in the fovea of the living primate. Recording from the intact eye of a living animal, we compare the patterns of activity evoked by the optogenetic actuator ChrimsonR with natural photoreceptor mediated stimulation in the same retinal ganglion cells. Optogenetic responses are recorded more than one year following administration of the therapy and two weeks after acute loss of photoreceptor input in the living animal. This in vivo imaging approach could be paired with any therapy to minimize the number of primates required to evaluate restored activity on the retinal level, while maximizing translational benefit by using an appropriate pre-clinical model of the human visual system.

Background/Aims Prior studies support an association between increased retinal venule diameter and elevated intracranial pressure (ICP). The purpose of this study was to test the hypothesis that retinal venule diameters decrease in association with long-term therapy for high ICP in subjects with idiopathic intracranial hypertension (IIH). Methods This is a retrospective analysis of multicentre randomised controlled trial data. Standardised procedures were used to measure area of optic nerve head elevation (ONHA) and diameters of 4 arterioles and 4 venules 2.7 mm from the optic disc centre on fundus photos collected at baseline and after 6 months of randomised treatment with placebo+diet or acetazolamide+diet in subjects participating in the IIH Treatment Trial (IIHTT) (n=115). Change in arteriole (Da) and venule (Dv) diameters from baseline to 6 months was studied as a function of IIH, haemodynamic and demographic variables. Results Dv decreased following 6 months of therapy (8.1 µm, 5.9%, p<0.0005) but Da did not change. Dv change was associated with ONHA change (p<0.0005, r=0.47) and this association persisted in multiple variable models. Conclusions Retinal venule diameter decreased, and arteriole diameter did not change in association with treatment for elevated ICP with a weight loss intervention and placebo or acetazolamide in IIHTT participants. Further study is needed to determine how retinal vessel measurements can be combined with other clinical observations to inform disease management.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

PURPOSE: To compare measurements of papilledema using fundus photography, optical coherence tomography (OCT), and Frisen score in patients with idiopathic intracranial hypertension (IIH). DESIGN: Retrospective, noncomparative analysis of randomized controlled trial data. METHODS: The Idiopathic Intracranial Hypertension Treatment Trial (IIHTT) evaluated weight management and treatment with acetazolamide compared with placebo in patients with IIH and mild visual loss. Among the 126 subjects in the IIHTT OCT substudy, fundus photographs and OCT scans of the optic disc were taken at baseline and at 6 and 12 months after enrollment. Trained readers scored each eye using a modified Frisen scale and measured the area of disc elevation. OCT scans assessed optic nerve head (ONH) volume. Correlations between volume and area were computed for both study and nonstudy eyes. RESULTS: Disc area and ONH volume were positively correlated at baseline (R-2 = 0.77 in study eyes, P < .001). Correlations between area and volume were similar in the treatment groups at baseline, but were weaker in the acetazolamide group compared with the placebo group at 6 months (R-2 = 0.25 vs R-2 = 0.76 in study eyes) and 12 months (R-2 = 0.19 vs R-2 = 0.65 in study eyes). At 6 and 12 months after enrollment, there was no consistent relationship between Frisen score, disc area, and ONH volumes in the acetazolamide group. CONCLUSION: Frisen score fails to reflect the photographic area and OCT volume of papilledema after treatment with acetazolamide. Clinicians should use caution when using the Frisen scale to monitor the effect of treatment on papilledema over time. (C) 2018 The Author(s). Published by Elsevier Inc.

Here we employ adaptive optics scanning light ophthalmoscopy (AOSLO) to assess cellular-scale changes in photoreceptor structure and function in a non-human primate model of retinal degeneration. The retinas of four macaques were subretinally injected with an adeno-associated viral construct designed to locally damage photoreceptors [1]. Before and after injection, OCT and fundus SLO imaging was performed. Post-injection fundus SLO reflectance images revealed regions of decreased intensity, suggesting retinal damage. OCT of affected regions showed a reduction of intensity in outer retinal layers, primarily in the interdigitation zone. In two retinas, photoreceptors in both affected and unaffected regions were imaged with AOSLO in three modalities (λ=730nm): confocal reflectance, two-photon autofluorescence, and multi-offset detection. Confocal reflectance was used to capture directly backscattered light primarily originating from the inner/outer segment boundary and outer segment tip [2]. Photoreceptors in affected regions exhibited reduced waveguiding, suggesting outer segment damage. In multi-offset detection, the confocal pinhole was displaced to capture multiply scattered light. Images from several aperture positions were combined to visualize inner segments [3], which were present in both affected and unaffected regions. Two-photon autofluorescence was used to excite all-trans-retinol and track its kinetics in response to light, which are indicative of retinoid production necessary for visual function and thus the functional state of the photoreceptors [4]. In affected photoreceptors, there was no detectable increase in autofluorescence at light onset as was observed in unaffected photoreceptors. Therefore, photoreceptor assessment using AOSLO and OCT is consistent with inhibition of retinoid production due to outer segment damage while inner segments are preserved. This model of retinal degeneration shows promise for preclinical testing of vision restoration methods.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Identify a reproducible measure of axial globe position (AGP) for multicenter studies on patients with thyroid eye disease (TED).This is a prospective, international, multicenter, observational study in which 3 types of AGP evaluation were examined: radiologic, clinical, and photographic. In this study, CT was the modality to which all other methods were compared. CT AGP was measured from an orthogonal line between the anterior lateral orbital rims to the cornea. All CT measurements were made at a single institution by 3 individual clinicians. Clinical evaluation was performed with exophthalmometry. Three clinicians from each clinical site assessed AGP with 3 different exophthalmometers and horizontal palpebral width using a ruler. Each physician made 3 separate measurements with each type of exophthalmometer not in succession. All photographic measurements were made at a single institution. AGP was measured from lateral photographs in which a standard marker was placed at the anterior lateral orbital rim. Horizontal and vertical palpebral fissure were measured from frontal photographs. Three trained readers measured 3 separate times not in succession. Exophthalmometry and photography method validity was assessed by agreement with CT (mean differences calculation, intraclass correlation coefficients [ICCs], Bland-Altman figures). Correlation between palpebral fissure and CT AGP was assessed with Pearson correlation. Intraclinician and interclinician reliability was evaluated using ICCs.Sixty-eight patients from 7 centers participated. CT mean AGP was 21.37 mm (15.96-28.90 mm) right and 21.22 mm (15.87-28.70 mm) left (ICC 0.996 and 0.995). Exophthalmometry AGP fell between 18 mm and 25 mm. Intraclinician agreement across exophthalmometers was ideal (ICC 0.948-0.983). Agreement between clinicians was greater than 0.85 for all upright exophthalmometry measurements. Photographic mean AGP was 20.47 mm (10.92-30.88 mm) right and 20.30 mm (8.61-28.72 mm) left. Intrareader and interreader agreement was ideal (ICC 0.991-0.989). All exophthalmometers' mean differences from CT ranged between -0.06 mm (±1.36 mm) and 0.54 mm (±1.61 mm); 95% confidence interval fell within 1 mm. Magnitude of AGP did not affect exophthalmometry validity. Oculus best estimated CT AGP but differences from other exophthalmometers were not clinically meaningful in upright measurements. Photographic AGP (right ICC = 0.575, left ICC = 0.355) and palpebral fissure do not agree with CT.Upright clinical exophthalmometry accurately estimates CT AGP in TED. AGP measurement was reliably reproduced by the same clinician and between clinicians at multiple institutions using the protocol in this study. These findings allow reliable measurement of AGP that will be of considerable value in future outcome studies.

As a biomarker of Alzheimer's disease (AD), we compare amyloid deposits in the neural retina in human and in the dog model of AD and their visibility in differing imaging modalities. Diseased eyes were from humans with a diagnosis of AD and age matched control eyes had no associated dementia or glaucoma diagnoses. Diseased and normal dogs were positive and negative for cognitive dysfunction syndrome respectively, diagnosed via cognitive impairment testing. Retinas were flat mounted and stained with fluorescent amyloid markers. Atomic force, confocal and polarization microscopies were used to image amyloid positive areas and negative areas of positive retinas and control retinas. 20 AD and 22 control human retinas and 6 positive and 7 negative dog retinas were analyzed. Retinas of 5 dogs, injected IV, were imaged in vivo in amyloid fluorescence and clinical optical coherence tomography (OCT). Atomic force and confocal microscopies localized amyloid deposits to the anterior neural retinal layers. Amyloid fluorescence occurred with similar high sensitivities and lower specificities in human and dog retinas. Retinal amyloid deposits showed contrast in polarized light, similar to that of pure amyloid beta deposits. The sensitivity and specificity of polarized light in identifying amyloid were similar to the more invasive fluorescence imaging. A fluorescence marker for amyloid crossed the blood retinal barrier in vivo in the dog model of AD and imaged presumed amyloid deposits which showed no contrast in OCT images. The presence of amyloid deposits with similar properties in the neural layers of human and dog retinas, in those positive for disease, strengthens their utility as a biomarker of AD. The specificity found is close to that of PET amyloid scans, suggesting that amyloid deposits are present in the retina prior to disease symptoms. The similarities between the deposits in the two species support the use of the dog model of AD. The resemblance of polarized light interaction between the retinal amyloid deposits and pure amyloid beta is consistent with the retinal deposits containing a high concentration of amyloid beta. Polarization imaging is promising for non-invasive imaging of retinal amyloid deposits as a biomarker of AD. These are false colour maps of selected polarization properties where dark blue indicates a value of zero and red indicates the highest value (1 for every property except retardance where the highest value is 180 deg). The top row shows a thioflavin-S amyloid positive deposit in a post-mortem human retina from an individual with a diagnosis of AD. The values of the polarization properties in the deposit differ significantly from the surrounding retina. The bottom row shows values for a pure Aβ deposit on glass. The polarization properties of the pure deposit are similar to those of the amyloid positive deposit in the retina. Polarization properties of amyloid deposits in human and dog retinas. Above and below on the left are the similar polarization properties of deposits in a human and a dog retina. The 4 false colour maps show different polarization properties. Colour scales are the same as in Figure 1. These polarization properties make the deposits visible against the surrounding retina. On the right are amyloid fluorescence images of the two deposits. Scale bars are 20 μm.

Purpose: Identify a reproducible measure of axial globe position (AGP) for multicenter studies on patients with thyroid eye disease (TED).Methods: This is a prospective, international, multicenter, observational study in which 3 types of AGP evaluation were examined: radiologic, clinical, and photographic. In this study, CT was the modality to which all other methods were compared. CT AGP was measured from an orthogonal line between the anterior lateral orbital rims to the cornea. All CT measurements were made at a single institution by 3 individual clinicians. Clinical evaluation was performed with exophthalmometry. Three clinicians from each clinical site assessed AGP with 3 different exophthalmometers and horizontal palpebral width using a ruler. Each physician made 3 separate measurements with each type of exophthalmometer not in succession. All photographic measurements were made at a single institution. AGP was measured from lateral photographs in which a standard marker was placed at the anterior lateral orbital rim. Horizontal and vertical palpebral fissure were measured from frontal photographs. Three trained readers measured 3 separate times not in succession. Exophthalmometry and photography method validity was assessed by agreement with CT (mean differences calculation, intraclass correlation coefficients [ICCs], Bland-Altman figures). Correlation between palpebral fissure and CT AGP was assessed with Pearson correlation. Intraclinician and interclinician reliability was evaluated using ICCs.Results: Sixty-eight patients from 7 centers participated. CT mean AGP was 21.37mm (15.96-28.90mm) right and 21.22mm (15.87-28.70mm) left (ICC 0.996 and 0.995). Exophthalmometry AGP fell between 18mm and 25mm. Intraclinician agreement across exophthalmometers was ideal (ICC 0.948-0.983). Agreement between clinicians was greater than 0.85 for all upright exophthalmometry measurements. Photographic mean AGP was 20.47mm (10.92-30.88mm) right and 20.30mm (8.61-28.72mm) left. Intrareader and interreader agreement was ideal (ICC 0.991-0.989). All exophthalmometers' mean differences from CT ranged between -0.06mm (1.36mm) and 0.54mm (+/- 1.61mm); 95% confidence interval fell within 1mm. Magnitude of AGP did not affect exophthalmometry validity. Oculus best estimated CT AGP but differences from other exophthalmometers were not clinically meaningful in upright measurements. Photographic AGP (right ICC = 0.575, left ICC = 0.355) and palpebral fissure do not agree with CT.Conclusions: Upright clinical exophthalmometry accurately estimates CT AGP in TED. AGP measurement was reliably reproduced by the same clinician and between clinicians at multiple institutions using the protocol in this study. These findings allow reliable measurement of AGP that will be of considerable value in future outcome studies.

Two-photon ophthalmoscopy has potential for in vivo assessment of function of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.