Mouse model of radiation retinopathy reveals vascular and neuronal injury.

PURPOSE: To characterize the neuronal and vascular pathology in vivo and in vitro in a mouse model of radiation retinopathy. METHODS: C57Bl/6J mice underwent cranial irradiation with 12 Gy and in vivo imaging by optical coherence tomography and of relative blood flow velocity by laser speckle flowgraphy for up to 3-6 months after irradiation. Retinal architecture, vascular density and leakage and apoptosis were analyzed by histology and immunohistochemistry before irradiation or at 10, 30, 240, and 365 days after treatment. RESULTS: The vascular density decreased in the plexiform layers starting at 30 days after irradiation. No impairment in retinal flow velocity was seen. Subtle perivascular leakage was present at 10 days, in particular in the outer plexiform layer. This corresponded to increased width of this layer. However, no significant change in the retinal thickness was detected by OCT-B scans. At 365 days after irradiation, the nuclear density was significantly reduced compared to baseline. Apoptosis was detected at 30 days and less prominent at 365 days. CONCLUSIONS: By histology, vascular leakage at 10 days was followed by increased neuronal apoptosis and loss of neuronal and vascular density. However, in vivo imaging approaches that are commonly used in human patients did not detect pathology in mice.

Quantitative Assessment of Subjective Symptoms and Corneal Sensitivity in Chronic Orbital Pain Patients.

PURPOSE: To objectively evaluate the subjective symptoms and characteristics of chronic orbital pain as well as to quantify sensitization of peripheral trigeminal nerves. METHODS: In this prospective cohort study, patients who previously showed a response to peripheral trigeminal nerve blocks for unilateral, idiopathic chronic orbital pain and healthy subjects completed validated questionnaires assessing headaches, neuropathic signs and symptoms, photophobia, and pain qualities. Corneal sensitivity was measured in both eyes for all subjects with a Cochet-Bonnet aesthesiometer. For pain patients, the full assessment protocol was repeated 2-4 weeks after the study injection, and corneal sensitivity was also measured 30 minutes postinjection. Outcomes assessed were headache, neuropathic pain, and photophobia scores; pain qualities; and corneal sensitivity. RESULTS: Six female chronic orbital pain patients (mean age 48.2 years) and 11 female controls (mean age 47.5) were included. The mean headache, neuropathic pain, and photophobia questionnaire scores were significantly higher for pain patients than for controls (p < 0.001). On sensory testing, 5 pain patients (83.3%) endorsed allodynia, and all 6 (100%) had hyperalgesia in the ipsilateral frontal nerve dermatome. No controls had allodynia or hyperalgesia. Corneal sensitivity was similar between eyes in pain patients and between groups. Questionnaire scores and corneal sensitivity did not change significantly after the injection. CONCLUSIONS: Chronic orbital pain patients have a measurable reduction in quality of life due to headaches and photophobia. The supraorbital and supratrochlear nerves are sensitized, resulting in cutaneous hypersensitivity in the corresponding dermatome, but corneal nerves have normal sensitivity.

Measuring hyperemic response to light flicker stimulus using continuous laser speckle flowgraphy in mice.

Alterations in neurovascular coupling have been associated with various ocular, cerebral, and systemic vascular disorders. In the eye, changes in vessel caliber by dynamic vessel analysis have been used to measure neurovascular coupling following a light flicker stimulus. Here, we present a new protocol for quantifying light-flicker induced hyperemia in the C57/Bl6J mouse retina using laser speckle flowgraphy (LSFG). Our protocol was adapted from protocols used in human subjects. By acquiring continuous time series data, we detected significant increase in blood flow. These responses are maintained with low variability over multiple imaging sessions, indicating these methods may be applied in serial studies of neurovascular coupling.

Reduced blood flow by laser speckle flowgraphy after 125I-plaque brachytherapy for uveal melanoma.

BACKGROUND: To determine whether reductions in retinal and choroidal blood flow measured by laser speckle flowgraphy are detected after 125I-plaque brachytherapy for uveal melanoma. METHODS: In a cross-sectional study, retinal and choroidal blood flow were measured using laser speckle flowgraphy in 25 patients after treatment with 125I-plaque brachytherapy for uveal melanoma. Flow was analyzed in the peripapillary region by mean blur rate as well as in the entire image area with a novel superpixel-based method. Relationships between measures were determined by Spearman correlation. RESULTS: Significant decreases in laser speckle blood flow were observed in both the retinal and choroidal vascular beds of irradiated, but not fellow, eyes. Overall, 24 of 25 patients had decreased blood flow compared to their fellow eye, including 5 of the 6 patients imaged within the first 6 months following brachytherapy. A significant negative correlation between blood flow and time from therapy was present. CONCLUSIONS: Decreases in retinal and choroidal blood flow by laser speckle flowgraphy were detected within the first 6 months following brachytherapy. Reduced retinal and choroidal blood flow may be an early indicator of microangiographic response to radiation therapy.

Clinical Correlation Between Vertical Gaze Palsy and Midbrain Volume in Progressive Supranuclear Palsy.

BACKGROUND: Supranuclear vertical gaze palsies and slowed vertical saccades are characteristic clinic features of progressive supranuclear palsy (PSP). The "hummingbird sign," reflective of midbrain atrophy, is a classic radiographic sign of PSP. Correlation between eye movement abnormalities and radiographic findings in PSP has been reported previously. However, due to the use of clinical criteria not commonly employed in neuro-ophthalmic practice and neuroimaging techniques that are not widely available, it remains unclear whether correlation between midbrain structure and characteristic ocular-motor disturbances can be helpful to neuro-ophthalmologists seeking to adjudicate difficult or unusual diagnostic cases. METHODS: Patients with a diagnosis of probable PSP according to Movement Disorders Society criteria were studied retrospectively. A neuroradiologist calculated brainstem volumes in enrolled participants and normal controls. Spearman correlations were used to correlate the extent of eye movement limitation as assessed by 2 neuro-ophthalmologists with brainstem volumes. RESULTS: Fourteen participants with PSP and 15 healthy controls with similar age and gender distribution were enrolled and evaluated retrospectively. All 14 participants with PSP had undergone MRIs. Midbrain atrophy significantly correlated with the PSP rating scale (P < 0.001). PSP patients had significantly reduced volumes in the midbrain (P -0.0026), tegmentum (0.0001), tectum (0.0001), and medulla (P = 0.0024) compared with normal controls. Notes documenting quantified ocular motor function were available in 7 of 14 participants with PSP. Midbrain atrophy significantly correlated with in the extent of upward gaze limitation (P = 0.03). CONCLUSIONS: The severity of upward gaze limitation correlates with the severity of midbrain atrophy in patients with PSP. Recognition of this correlation may help to adjudicate diagnostic dilemmas and guide further evaluation.

Peripheral Trigeminal Nerve Blocks for Chronic Orbital Pain: Clinical Features and Outcomes.

PURPOSE: To characterize chronic orbital pain in patients who benefitted from peripheral trigeminal nerve blocks and to explore the relationship between pain etiologies and phenotypes, injection attributes, and positive response to treatment. METHODS: In this single-center retrospective descriptive study, patients who underwent peripheral trigeminal nerve blocks for chronic orbital pain from November 2016 to May 2021 were selected. Data reviewed included inciting factors, neuropathic symptoms of orbital pain, injection composition (anesthetic alone versus anesthetic + dexamethasone), and corneal epitheliopathy grades. Primary outcomes assessed were response to injection, duration of injection effectiveness, and overall treatment efficacy. Associations between subgroups of chronic orbital pain, injection attributes, and treatment outcomes were examined. RESULTS: Nineteen patients who underwent a total of 94 peripheral trigeminal nerve blocks for chronic orbital pain were included. During a mean follow-up period of 2.4 years after initial injection (range 7 days-4.6 years), 16 (84.2%) patients achieved either partial or complete improvement. Ocular versus nonocular origin of orbital pain or the presence of neuropathic sensory characteristics was not associated with a treatment outcome. Injections containing dexamethasone had a lower positive efficacy (relative risk, 0.88; 95% CI, 0.81-0.97) and no statistically significant association with prolonged effect. Twenty-nine (50.9%) of the 57 injections for which effect duration was recorded produced a response lasting greater than 6 weeks. CONCLUSIONS: Modulation of trigeminal afferent nerve activity with peripheral trigeminal nerve blocks containing anesthetic with or without dexamethasone may be a promising treatment strategy for chronic orbital pain of diverse etiologies and phenotypes.

Optical Coherence Tomography Neuro-Toolbox for the Diagnosis and Management of Papilledema, Optic Disc Edema, and Pseudopapilledema.

BACKGROUND: Distinguishing optic disc edema from pseudopapilledema is a common, sometimes challenging clinical problem. Advances in spectral-domain optical coherence tomography (SD-OCT) of the optic nerve head (ONH) has proven to be a cost effective, noninvasive, outpatient procedure that may help. At its core are tools that quantify the thickness of the retinal nerve fiber layer (RNFL) and ganglion cell-inner plexiform layer (GC-IPL). The SD-OCT also provides a set of tools that may be qualitatively interpreted in the same way that we read an MRI. They include the transverse axial, en face, and circular tomogram. Our goal is to describe a practical office-based set of tools using SD-OCT in the diagnosis and monitoring of papilledema, optic disc edema, and pseudopapilledema. EVIDENCE ACQUISITION: Searches on PubMed were performed using combinations of the following key words: OCT, papilledema, pseudopapilledema, optic disc drusen, retinal folds (RF), and choroidal folds (CF). RESULTS: The principal elements of SD-OCT analysis of the ONH are the RNFL and GC-IPL thickness; however, these metrics have limitations when swelling is severe. Qualitative interpretation of the transverse axial SD-OCT aids in assessing peripapillary shape that may help distinguish papilledema from pseudopapilledema, evaluate atypical optic neuropathies, diagnose shunt failures, and identify outer RF and CF. There is a consensus that the SD-OCT is the most sensitive way of identifying buried optic disc drusen. En face SD-OCT is especially effective at detecting peripapillary wrinkles and outer retinal creases, both of which are common and distinctive signs of optic disc edema that rule out pseudopapilledema. Mechanically stressing the ONH in the adducted eye position, in patients with papilledema, may expose folds and peripapillary deformations that may not be evident in primary position. We also discuss how to optimize the acquisition and registration of SD-OCT images. CONCLUSIONS: The SD-OCT is not a substitute for a complete history and a careful examination. It is, however, a convenient ancillary test that aids in the diagnosis and management of papilledema, optic disc edema, and pseudopapilledema. It is particularly helpful in monitoring changes over the course of time and distinguishing low-grade papilledema from buried drusen. The application of the SD-OCT toolbox depends on optimizing the acquisition of images, understanding its limitations, recognizing common artifacts, and accurately interpreting images in the context of both history and clinical findings.

Upper Eyelid Response to Topical 0.5% Apraclonidine.

PURPOSE: To describe the change in upper eyelid position in a self-reportedly normal population after the administration of topical 0.5% apraclonidine in each eye. METHODS: One hundred self-reportedly normal subjects received a 1-time administration of topical 0.5% apraclonidine in each eye. Digital photographs were taken at baseline and then 30 and 45 minutes following apraclonidine instillation. Marginal reflex distance 1 was determined via image analysis of acquired digital photographs (image-derived measurements are given the prefix "i" in this study). The horizontal corneal diameter was used as a constant measurement scale in each photograph. RESULTS: The mean increase in i-marginal reflex distance 1 post-administration of 0.5% apraclonidine was +0.70 ± 0.60 mm (range, -0.94 to +2.66 mm) after 30 minutes and +0.68 ± 0.59 mm (range, -0.69 to +2.54 mm) after 45 minutes. Of the 200 total eyelids in 100 subjects, 181 (90.5%) had an increase in i-marginal reflex distance 1 at 30 minutes. Of the 100 subjects, 85 (85%) had a bilateral increase in i-marginal reflex distance 1, 4 (4%) had a bilateral decrease, and 11 (11%) had a unilateral increase with a contralateral decrease. CONCLUSIONS: Given its predominant small-amplitude upper eyelid elevating effect, topical apraclonidine may be a useful off-label alternative treatment for mild upper eyelid ptosis and in eyelid asymmetry due to eyelid retraction through use in the contralateral eye.

Optical Coherence Angiographic Demonstration of Retinal Changes From Chronic Optic Neuropathies.

Glaucoma causes a decrease in peripapillary perfused capillary density on optical coherence tomography (OCT) angiography. However, other chronic optic neuropathies have not been explored with OCT angiography to see if these changes were specific to glaucoma. The authors evaluated OCT angiography in 10 patients who suffered various kinds of chronic optic neuropathies, including optic neuritis and ischaemic optic neuropathy, and found that all optic neuropathies showed a decrease in peripapillary vessel density on OCT angiography, regardless of the aetiology of the optic neuropathy. The peripapillary vessel loss on OCT angiography correlated well with the areas of retinal nerve fibre layer thinning seen on OCT.

Avoiding Clinical Misinterpretation and Artifacts of Optical Coherence Tomography Analysis of the Optic Nerve, Retinal Nerve Fiber Layer, and Ganglion Cell Layer.

BACKGROUND: Optical coherence tomography (OCT) has become an important tool for diagnosing optic nerve disease. The structural details and reproducibility of OCT continues to improve with further advances in technology. However, artifacts and misinterpretation of OCT can lead to clinical misdiagnosis of diseases if they go unrecognized. EVIDENCE ACQUISITION: A literature review using PubMed combined with clinical and research experience. RESULTS: We describe the most common artifacts and errors in interpretation seen on OCT in both optic nerve and ganglion cell analyses. We provide examples of the artifacts, discuss the causes, and provide methods of detecting them. In addition, we discuss a systematic approach to OCT analysis to facilitate the recognition of artifacts and to avoid clinical misinterpretation. CONCLUSIONS: While OCT is invaluable in diagnosing optic nerve disease, we need to be cognizant of the artifacts that can occur with OCT. Failure to recognize some of these artifacts can lead to misdiagnoses and inappropriate investigations.

Effect of enriching the diet with menhaden oil or daily treatment with resolvin D1 on neuropathy in a mouse model of type 2 diabetes.

The purpose of this study was to determine the effect of supplementing the diet of a mouse model of type 2 diabetes with menhaden (fish) oil or daily treatment with resolvin D1 on diabetic neuropathy. The end points evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and the retinal ganglion cell complex thickness. Menhaden oil is a natural source for n-3 polyunsaturated fatty acids, which have been shown to have beneficial effects in other diseases. Resolvin D1 is a metabolite of docosahexaenoic acid and is known to have anti-inflammatory and neuroprotective properties. To model type 2 diabetes, mice were fed a high-fat diet for 8 wk followed by a low dosage of streptozotocin. After 8 wk of hyperglycemia, mice in experimental groups were treated for 6 wk with menhaden oil in the diet or daily injections of 1 ng/g body wt resolvin D1. Our findings show that menhaden oil or resolvin D1 did not improve elevated blood glucose, HbA1C, or glucose utilization. Untreated diabetic mice were thermal hypoalgesic, had reduced motor and sensory nerve conduction velocities, had decreased innervation of the cornea and skin, and had thinner retinal ganglion cell complex. These end points were significantly improved with menhaden oil or resolvin D1 treatment. Exogenously, resolvin D1 stimulated neurite outgrowth from primary cultures of dorsal root ganglion neurons from normal mice. These studies suggest that n-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for diabetic neuropathy.

Effect of diet-induced obesity or type 1 or type 2 diabetes on corneal nerves and peripheral neuropathy in C57Bl/6J mice.

We determined the impact diet-induced obesity (DIO) and types 1 and 2 diabetes have on peripheral neuropathy with emphasis on corneal nerve structural changes in C57Bl/6J mice. Endpoints examined included nerve conduction velocity, response to thermal and mechanical stimuli and innervation of the skin and cornea. DIO mice and to a greater extent type 2 diabetic mice were insulin resistant. DIO and both types 1 and 2 diabetic mice developed motor and sensory nerve conduction deficits. In the cornea of DIO and type 2 diabetic mice there was a decrease in sub-epithelial corneal nerves, innervation of the corneal epithelium, and corneal sensitivity. Type 1 diabetic mice did not present with any significant changes in corneal nerve structure until after 20 weeks of hyperglycemia. DIO and type 2 diabetic mice developed corneal structural damage more rapidly than type 1 diabetic mice although hemoglobin A1 C values were significantly higher in type 1 diabetic mice. This suggests that DIO with or without hyperglycemia contributes to development and progression of peripheral neuropathy and nerve structural damage in the cornea.

Homonymous Ganglion Cell Layer Thinning After Isolated Occipital Lesion: Macular OCT Demonstrates Transsynaptic Retrograde Retinal Degeneration.

A 48-year-old man was examined 24 months after medial and surgical treatment of an isolated well-circumscribed right occipital lobe abscess. An asymptomatic residual left homonymous inferior scotoma was present. Fundus examination revealed temporal pallor of both optic discs, and optical coherence tomography (OCT) revealed mild temporal loss of retinal nerve fiber layer in both eyes. No relative afferent pupillary defect was present. Assessment of the retinal ganglion cell layer demonstrated homonymous thinning in a pattern corresponding to the homonymous visual field loss. There were no abnormalities of the lateral geniculate nuclei or optic tracts on review of the initial brain computed tomography and follow-up magnetic resonance imaging. We believe our patient showed evidence of transsynaptic retrograde degeneration after an isolated right occipital lobe lesion, and the homonymous neuronal loss was detected on OCT by assessing the retinal ganglion cell layer.

Effect of glycemic control on corneal nerves and peripheral neuropathy in streptozotocin-induced diabetic C57Bl/6J mice.

We sought to determine the impact that duration of hyperglycemia and control has on corneal nerve fiber density in relation to standard diabetic neuropathy endpoints. Control and streptozotocin-diabetic C57Bl/6J mice were analyzed after 4, 8, 12, and 20 weeks. For the 20-week time point, five groups of mice were compared: control, untreated diabetic, and diabetic treated with insulin designated as having either poor glycemic control, good glycemic control, or poor glycemic control switched to good glycemic control. Hyperglycemia was regulated by use of insulin-releasing pellets. Loss of corneal nerves in the sub-epithelial nerve plexus or corneal epithelium progressed slowly in diabetic mice requiring 20 weeks to reach statistical significance. In comparison, slowing of motor and sensory nerve conduction velocity developed rapidly with significant difference compared with control mice observed after 4 and 8 weeks of hyperglycemia, respectively. In diabetic mice with good glycemic control, average blood glucose levels over the 20-week experimental period were lowered from 589 ± 2 to 251 ± 9 mg/dl. All diabetic neuropathy endpoints examined were improved in diabetic mice with good glycemic control compared with untreated diabetic mice. However, good control of blood glucose was not totally sufficient in preventing diabetic neuropathy.

Diagnostic features of retinal nerve fiber layer rotation in skew deviation using optical coherence tomography.

A 41-year-old woman with skew deviation had cyclotorsion of both eyes. This resulted in a falsely low probability plot of retinal nerve fiber layer thickness in adjacent clock hours on optical coherence tomography (OCT) due to displacement of the retinal nerve fiber layer peaks. Ocular cyclotorsion may cause misinterpretation of OCT probability plots. OCT retinal nerve fiber layer plots also may be used to objectively quantify the degree of ocular cyclotorsion.

Visualization of Optic Nerve Structural Patterns in Papilledema Using Deep Learning Variational Autoencoders.

Purpose: To visualize and quantify structural patterns of optic nerve edema encountered in papilledema during treatment. Methods: A novel bi-channel deep-learning variational autoencoder (biVAE) model was trained using 1498 optical coherence tomography (OCT) scans of 125 subjects over time from the Idiopathic Intracranial Hypertension Treatment Trial (IIHTT) and 791 OCT scans of 96 control subjects from the University of Iowa. An independent test dataset of 70 eyes from 70 papilledema subjects was used to evaluate the ability of the biVAE model to quantify and reconstruct the papilledema spatial patterns from input OCT scans using only two variables. Results: The montage color maps of the retinal nerve fiber layer (RNFL) and total retinal thickness (TRT) produced by the biVAE model provided an organized visualization of the variety of morphological patterns of optic disc edema (including differing patterns at similar thickness levels). Treatment effects of acetazolamide versus placebo in the IIHTT were also demonstrated in the latent space. In image reconstruction, the mean signed peripapillary retinal nerve fiber layer thickness (pRNFLT) difference ± SD was -0.12 ± 17.34 µm, the absolute pRNFLT difference was 13.68 ± 10.65 µm, and the RNFL structural similarity index reached 0.91 ± 0.05. Conclusions: A wide array of structural patterns of papilledema, integrating the magnitude of disc edema with underlying disc and retinal morphology, can be quantified by just two latent variables. Translational Relevance: A biVAE model encodes structural patterns, as well as the correlation between channels, and may be applied to visualize individuals or populations with papilledema throughout treatment.

Hybrid deep learning and optimal graph search method for optical coherence tomography layer segmentation in diseases affecting the optic nerve.

Accurate segmentation of retinal layers in optical coherence tomography (OCT) images is critical for assessing diseases that affect the optic nerve, but existing automated algorithms often fail when pathology causes irregular layer topology, such as extreme thinning of the ganglion cell-inner plexiform layer (GCIPL). Deep LOGISMOS, a hybrid approach that combines the strengths of deep learning and 3D graph search to overcome their limitations, was developed to improve the accuracy, robustness and generalizability of retinal layer segmentation. The method was trained on 124 OCT volumes from both eyes of 31 non-arteritic anterior ischemic optic neuropathy (NAION) patients and tested on three cross-sectional datasets with available reference tracings: Test-NAION (40 volumes from both eyes of 20 NAION subjects), Test-G (29 volumes from 29 glaucoma subjects/eyes), and Test-JHU (35 volumes from 21 multiple sclerosis and 14 control subjects/eyes) and one longitudinal dataset without reference tracings: Test-G-L (155 volumes from 15 glaucoma patients/eyes). In the three test datasets with reference tracings (Test-NAION, Test-G, and Test-JHU), Deep LOGISMOS achieved very high Dice similarity coefficients (%) on GCIPL: 89.97±3.59, 90.63±2.56, and 94.06±1.76, respectively. In the same context, Deep LOGISMOS outperformed the Iowa reference algorithms by improving the Dice score by 17.5, 5.4, and 7.5, and also surpassed the deep learning framework nnU-Net with improvements of 4.4, 3.7, and 1.0. For the 15 severe glaucoma eyes with marked GCIPL thinning (Test-G-L), it demonstrated reliable regional GCIPL thickness measurement over five years. The proposed Deep LOGISMOS approach has potential to enhance precise quantification of retinal structures, aiding diagnosis and treatment management of optic nerve diseases.

Radiation effects on retinal layers revealed by OCT, OCT-A, and perimetry as a function of dose and time from treatment.

Optical coherence tomography (OCT) has become a key method for diagnosing and staging radiation retinopathy, based mainly on the presence of fluid in the central macula. A robust retinal layer segmentation method is required for identification of the specific layers involved in radiation-induced pathology in individual eyes over time, in order to determine damage driven by radiation injury to the microvessels and to the inner retinal neurons. Here, we utilized OCT, OCT-angiography, visual field testing, and patient-specific dosimetry models to analyze abnormal retinal layer thickening and thinning relative to microvessel density, visual function, radiation dose, and time from radiotherapy in a cross-sectional cohort of uveal melanoma patients treated with 125I-plaque brachytherapy. Within the first 24 months of radiotherapy, we show differential thickening and thinning of the two inner retinal layers, suggestive of microvessel leakage and neurodegeneration, mostly favoring thickening. Four out of 13 eyes showed decreased inner retinal capillary density associated with a corresponding normal inner retinal thickness, indicating early microvascular pathology. Two eyes showed the opposite: significant inner retinal layer thinning and normal capillary density, indicating early neuronal damage preceding a decrease in capillary density. At later time points, inner retinal thinning becomes the dominant pathology and correlates significantly with decreased vascularity, vision loss, and dose to the optic nerve. Stable multiple retinal layer segmentation provided by 3D graph-based methods aids in assessing the microvascular and neuronal response to radiation, information needed to target therapeutics for radiation retinopathy and vision loss.

Effectiveness of averaging strategies to reduce variance in retinal nerve fibre layer thickness measurements using spectral-domain optical coherence tomography.

BACKGROUND: Automated detection of subtle changes in peripapillary retinal nerve fibre layer thickness (RNFLT) over time using optical coherence tomography (OCT) is limited by inherent image quality before layer segmentation, stabilization of the scan on the peripapillary retina and its precise placement on repeated scans. The present study evaluates image quality and reproducibility of spectral domain (SD)-OCT comparing different rates of automatic real-time tracking (ART). METHODS: Peripapillary RNFLT was measured in 40 healthy eyes on six different days using SD-OCT with an eye-tracking system. Image brightness of OCT with unaveraged single frame B-scans was compared to images using ART of 16 B-scans and 100 averaged frames. Short-term and day-to-day reproducibility was evaluated by calculation of intraindividual coefficients of variation (CV) and intraclass correlation coefficients (ICC) for single measurements as well as for seven repeated measurements per study day. RESULTS: Image brightness, short-term reproducibility, and day-to-day reproducibility were significantly improved using ART of 100 frames compared to one and 16 frames. Short-term CV was reduced from 0.94 ± 0.31 % and 0.91 ± 0.54 % in scans of one and 16 frames to 0.56 ± 0.42 % in scans of 100 averaged frames (P ≤ 0.003 each). Day-to-day CV was reduced from 0.98 ± 0.86 % and 0.78 ± 0.56 % to 0.53 ± 0.43 % (P ≤ 0.022 each). The range of ICC was 0.94 to 0.99. Sample size calculations for detecting changes of RNFLT over time in the range of 2 to 5 µm were performed based on intraindividual variability. CONCLUSION: Image quality and reproducibility of mean peripapillary RNFLT measurements using SD-OCT is improved by averaging OCT images with eye-tracking compared to unaveraged single frame images. Further improvement is achieved by increasing the amount of frames per measurement, and by averaging values of repeated measurements per session. These strategies may allow a more accurate evaluation of RNFLT reduction in clinical trials observing optic nerve degeneration.