Hyperglycemia Promotes Mitophagy and Thereby Mitigates Hyperglycemia-Induced Damage.

The observation that diabetic retinopathy (DR) typically takes decades to develop suggests the existence of an endogenous system that protects from diabetes-induced damage. To investigate the existance of such a system, primary human retinal endothelial cells were cultured in either normal glucose (5 mmol/L) or high glucose (30 mmol/L; HG). Prolonged exposure to HG was beneficial instead of detrimental. Although tumor necrosis factor-α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 was unaffected after 1 day of HG, it waned as the exposure to HG was extended. Similarly, oxidative stress-induced death decreased with prolonged exposure to HG. Furthermore, mitochondrial functionality, which was compromised by 1 day of HG, was improved by 10 days of HG, and this change required increased clearance of damaged mitochondria (mitophagy). Finally, antagonizing mitochondrial dynamics compromised the cells' ability to endure HG: susceptibility to cell death increased, and basal barrier function and responsiveness to vascular endothelial growth factor deteriorated. These observations indicate the existence of an endogenous system that protects human retinal endothelial cells from the deleterious effects of HG. Hyperglycemia-induced mitochondrial adaptation is a plausible contributor to the mechanism responsible for the delayed onset of DR; loss of hyperglycemia-induced mitochondrial adaptation may set the stage for the development of DR.

Bone-marrow mesenchymal stem-cell administration significantly improves outcome after retinal ischemia in rats.

PURPOSE: Ischemia-associated retinal degeneration is one of the leading causes of vision loss, and to date, there are no effective treatment options. We hypothesized that delayed injection of bone-marrow stem cells (BMSCs) 24 h after the onset of ischemia could effectively rescue ischemic retina from its consequences, including apoptosis, inflammation, and increased vascular permeability, thereby preventing retinal cell loss. METHODS: Retinal ischemia was induced in adult Wistar rats by increasing intraocular pressure (IOP) to 130-135 mmHg for 55 min. BMSCs harvested from rat femur were injected into the vitreous 24 h post-ischemia. Functional recovery was assessed 7 days later using electroretinography (ERG) measurements of the a-wave, b-wave, P2, scotopic threshold response (STR), and oscillatory potentials (OP). The retinal injury and anti-ischemic effects of BMSCs were quantitated by measuring apoptosis, autophagy, inflammatory markers, and retinal-blood barrier permeability. The distribution and fate of BMSC were qualitatively examined using real-time fundus imaging, and retinal flat mounts. RESULTS: Intravitreal delivery of BMSCs significantly improved recovery of the ERG a- and b-waves, OP, negative STR, and P2, and attenuated apoptosis as evidenced by decreased TUNEL and caspase-3 protein levels. BMSCs significantly increased autophagy, decreased inflammatory mediators (TNF-α, IL-1ß, IL-6), and diminished retinal vascular permeability. BMSCs persisted in the vitreous and were also found within ischemic retina. CONCLUSIONS: Taken together, our results indicate that intravitreal injection of BMSCs rescued the retina from ischemic damage in a rat model. The mechanisms include suppression of apoptosis, attenuation of inflammation and vascular permeability, and preservation of autophagy.

Induction of autophagy in rats upon overexpression of wild-type and mutant optineurin gene.

BACKGROUND: Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis. It has been reported previously that in cultured RGC5 cells, the turnover of endogenous optineurin involves mainly the ubiquitin-proteasome pathway (UPP). When optineurin is upregulated or mutated, the UPP function is compromised as evidenced by a decreased proteasome ß5 subunit (PSMB5) level and autophagy is induced for clearance of the optineurin protein. RESULTS: Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP) only, GFP-tagged wild-type and Glu50Lys (E50K) mutated optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Following intravitreal injections, eyes that received optineurin vectors exhibited retinal thinning, as well as RGC and axonal loss compared to GFP controls. By immunostaining and Western blotting, the level of PSMB5 and autophagic substrate degradation marker p62 was reduced, and the level of autophagic marker microtubule associated protein 1 light chain 3 (LC3) was enhanced. The UPP impairment and autophagy induction evidently occurred in vivo as in vitro. The optineurin level, RGC and axonal counts, and apoptosis in AAV2-E50K-GFP-injected rat eyes were averted to closer to normal limits after treatment with rapamycin, an autophagic enhancer. CONCLUSIONS: The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.

Feasibility of assessment of conjunctival microvascular hemodynamics in unilateral ischemic stroke.

Since the internal carotid artery supplies blood to both the eye and the brain, ocular microvascular hemodynamics can be altered due to ischemic stroke. The purpose of the current study was to establish the feasibility of conjunctival microcirculation imaging for detection of inter-ocular differences in microvascular hemodynamics in subjects with unilateral ischemic stroke. Conjunctival microcirculation imaging was performed in both eyes of 15 healthy control subjects and 12 subjects following unilateral ischemic stroke. Diameter and axial blood velocity were measured in multiple conjunctival venules of each eye. A two-way repeated measures analysis of variance was performed to determine the effects of stroke (control vs. stroke) and side of stroke (ipsilateral vs. contralateral) on conjunctival diameter and axial blood velocity. There was no significant main effect of stroke on conjunctival diameter (P=0.7) or conjunctival axial blood velocity (P=0.9). There was no significant main effect of side of stroke on conjunctival diameter (P=0.8), but there was a significant main effect of side of stroke on conjunctival axial blood velocity (P=0.02). There was a significant interaction effect between stroke and side of stroke (P=0.04), indicating that conjunctival axial blood velocity was lower in ipsilateral eyes than in contralateral eyes of stroke subjects. Conjunctival axial blood velocity and internal carotid artery blood velocity were correlated in stroke subjects (r=0.75, P=0.01, N=10). Conjunctival microcirculation imaging is a feasible method to detect inter-ocular differences in microvascular hemodynamics in subjects with unilateral ischemic stroke.

Neural constraints on visual acuity in proliferative diabetic retinopathy.

PURPOSE: Visual acuity (VA) in normally sighted individuals is highly correlated with equivalent intrinsic blur, a measure of the amount of blur within the visual system that is generated by optical and neural sources. This study assessed the extent to which VA, equivalent intrinsic blur, optical blur, and neural blur are abnormal in subjects with proliferative diabetic retinopathy (PDR) and characterized the relationships among these parameters. METHODS: Best-corrected VA of 10 subjects with PDR (ages 25 to 68) and 10 normally sighted individuals (ages 46 to 63) was measured for tumbling E optotypes. The Es were either unblurred or blurred through convolution with Gaussian functions of different widths. Values of equivalent intrinsic blur (σ(int)) and unblurred VA (MAR0) were derived using a standard model. Optical blur (σ(opt)), a measure of blur generated by higher-order aberrations, was quantified using Shack-Hartmann aberrometry. An index of neural blur (η) was defined as 1--σ(opt)/σ(int), which represents the remaining blur once the contributions of σ(opt) to σ(int) have been accounted for. RESULTS: Log MAR0 and log σ(int) were correlated significantly (r = 0.98, p < 0.05) for the PDR subjects and the values of these parameters ranged from normal to more than a factor of 2 above the upper limit of normal. In comparison, log MAR measured for the most blurred E was elevated by a relatively small amount for all PDR subjects and was not correlated significantly with log σ(int) (r = 0.40, p = 0.25). MAR0, σ(int), and η differed significantly between the PDR subjects and the controls (all p < 0.05) but σ(opt) did not (p = 0.50). CONCLUSIONS: Subjects with PDR and VA loss had higher than normal equivalent intrinsic blur that resulted primarily from neural blur elevations, suggesting that neural blur is an important factor that limits VA in these patients.

Wavefront error correction with adaptive optics in diabetic retinopathy.

PURPOSE: To determine the effects of diabetic retinopathy (DR), increased foveal thickness (FT), and adaptive optics (AO) on wavefront aberrations and Shack-Hartmann (SH) image quality. METHODS: Shack-Hartmann aberrometry and wavefront error correction were performed with a bench-top AO retinal imaging system in 10 healthy control and 19 DR subjects. Spectral domain optical coherence tomography was performed and central FT was measured. Based on the FT data in the control group, subjects in the DR group were categorized into two subgroups: those with normal FT and those with increased FT. Shack-Hartmann image quality was assessed based on spot areas, and high-order (HO) root mean square (RMS) and total RMS were calculated. RESULTS: There was a significant effect of DR on HO and total RMS (p = 0.01), and RMS decreased significantly after AO correction (p < 0.001). Shack-Hartmann spot area was significantly affected by DR (p < 0.001), but it did not change after AO correction (p = 0.6). High-order RMS, total RMS, and SH spot area were higher in DR subjects both before and after AO correction. In DR subgroups, HO and total RMS decreased significantly after AO correction (p < 0.001), whereas the effect of increased FT on HO and total RMS was not significant (p ≥ 0.7). There were no significant effects of increased FT and AO on SH spot area (p = 0.9). CONCLUSIONS: Diabetic retinopathy subjects had higher wavefront aberrations and less compact SH spots, likely attributable to pathological changes in the ocular optics. Wavefront aberrations were significantly reduced by AO, although AO performance was suboptimal in DR subjects as compared with control subjects.

En face spectral-domain optical coherence tomography outer retinal analysis and relation to visual acuity.

PURPOSE: To describe a method of en face visualization and quantification of the photoreceptor inner segment/outer segment junction area, using spectral-domain optical coherence tomography, and association with visual acuity. METHODS: Case series of 74 eyes in 53 patients. Central 1-mm and 400-µm en face areas were analyzed with a computer algorithm. RESULTS: The presence or absence of inner segment/outer segment junction was visible on both spectral-domain optical coherence tomography en face and retinal cross sections. Thirty eyes (40.6%) had no retinal pathology and an average logMAR visual acuity of 0.116. Twenty-five eyes (33.8%) had intraretinal edema, with visual acuity of 0.494. Nineteen eyes had nonneovascular age-related macular degeneration (dry age-related macular degeneration, 25.6%), with visual acuity of 0.392. In all eyes, central 1-mm and 400-µm en face areas were 58.3 ± 25.0% and 56.4 ± 26.0%, which showed significant correlation with visual acuity (Pearson correlation, r = -0.66 and -0.56, both P < 0.001). This correlation was greater than correlation of visual acuity with central subfield thickness (r = 0.39, P < 0.001), macular volume (r = 0.36, P = 0.002), and average macular thickness (r = 0.37, P = 0.001). However, no variables were significantly correlated with dry age-related macular degeneration eyes. CONCLUSION: Central en face inner segment/outer segment junction areas are significantly correlated with visual acuity in most eyes. This may correlate better with visual acuity than other spectral-domain optical coherence tomography values, as a reflection of photoreceptor integrity. Dry age-related macular degeneration may disrupt the plane used to formulate the en face display. Advancements in spectral-domain optical coherence tomography may provide routine en face visualization analysis.

Contributions of optical and non-optical blur to variation in visual acuity.

PURPOSE: To determine the relative contributions of optical and non-optical sources of intrinsic blur to variations in visual acuity (VA) among normally sighted subjects. METHODS: Best-corrected VA of 16 normally sighted subjects was measured using briefly presented (59 ms) tumbling E optotypes that were either unblurred or blurred through convolution with Gaussian functions of different widths. A standard model of intrinsic blur was used to estimate each subject's equivalent intrinsic blur (σint) and VA for the unblurred tumbling E (MAR0). For 14 subjects, a radially averaged optical point spread function due to higher-order aberrations was derived by Shack-Hartmann aberrometry and fit with a Gaussian function. The standard deviation of the best-fit Gaussian function defined optical blur (σopt). An index of non-optical blur (η) was defined as 1 - σopt/σint. A control experiment was conducted on five subjects to evaluate the effect of stimulus duration on MAR0 and σint. RESULTS.: The logarithm of the minimum angle of resolution (logMAR0) for the briefly presented E was correlated significantly with log σint (r = 0.95, p < 0.01), consistent with previous work. However, logMAR0 was not correlated significantly with log σopt (r = 0.46, p = 0.11). For subjects with logMAR0 equivalent to ~20/20 or better, logMAR0 was independent of log η, whereas for subjects with larger logMAR0 values, logMAR0 was proportional to log η. The control experiment showed a statistically significant effect of stimulus duration on logMAR0 (p < 0.01) but a non-significant effect on σint (p = 0.13). CONCLUSIONS: The relative contributions of optical and non-optical blur to VA varied among the subjects and were related to the subject's VA. Evaluating optical and non-optical blur may be useful for predicting changes in VA following procedures that improve the optics of the eye in patients with both optical and non-optical sources of VA loss.

VEGFR2 Trafficking by KIF13B Is a Novel Therapeutic Target for Wet Age-Related Macular Degeneration.

Purpose: Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 are promising therapeutic targets for wet age-related macular degeneration (AMD). As a topically applicable option, we developed the peptide KAI to selectively interfere with VEGFR2 trafficking to the cell surface where it receives VEGF. This study sought to determine the efficacy of KAI in the mouse model of choroidal neovascularization (CNV). Methods: The specificity of KAI was tested by surface plasmon resonance. The drug delivery was analyzed by cryosection and the ELISA after treatment of KAI eyedrop to the mouse eyes. For the laser-induced CNV model, mice with laser-induced ruptures in Bruch's membrane received daily treatment of KAI eyedrop or control peptide. The other groups of mice received intravitreal injection of anti-VEGF or IgG control. After two weeks, CNV was quantified and compared. Results: First, we showed the specificity and high affinity of KAI to VEGFR2. Next, biodistribution revealed successful delivery of KAI eyedrop to the back of the mouse eyes. KAI significantly reduced the disease progression in laser-induced CNV. The comparison with current therapy suggests that KAI eyedrop is as effective as current therapy to prevent CNV in wet AMD. Moreover, the genetic deletion of a kinesin KIF13B, which mediates VEGFR2 trafficking to the cell surface, confirmed the pivotal role of KIF13B in disease progression of wet AMD and neovascularization from choroidal vessels. Conclusions: Taken together, pharmacologic inhibition and genetic deletion complementarily suggest the therapeutic possibility of targeting VEGFR2 trafficking to inhibit pathological angiogenesis in wet AMD.

Correlation between microaneurysms and retinal thickness in diabetic macular edema.

PURPOSE: Microaneurysms commonly are believed to be related causally to retinal thickening in diabetic retinopathy, especially by leaking. The hypothesis that thicker areas of retina in diabetic retinopathy have more microaneurysms per unit area than areas that are not as thick was tested. METHODS: Retinal thickness analysis was performed with a prototype instrument for 27 eyes of 27 diabetic patients and 22 normal eyes of 22 healthy subjects. Maps of retinal thickness were created, and microaneurysms were counted in zones having four levels of retinal thickness. RESULTS: There was no increase in either total microaneurysms or apparent leaking microaneurysms per unit area with increasing levels of retinal thickness (P = 0.77 and 0.87, respectively). CONCLUSION: Some microaneurysms may not cause thickening, or other factors may contribute to retinal thickening in diabetic retinopathy. The results may have implications on the pathogenesis of diabetic macular edema.

A method for three-dimensional imaging of the retina in human eyes.

BACKGROUND AND OBJECTIVES: An optical system for three-dimensional imaging of the retinal tissue in human eyes is described. PATIENTS AND METHODS: A laser beam was projected at an oblique angle on the retina and scanned to acquire 40 optical section images in a 1.0 x 1.5 mm retinal area. Because the incident laser beam was not coaxial with the viewing system, structures at various retinal depths appeared laterally displaced according to their depth location on the optical section image. The optical section images were segmented to construct a series of en face retinal images, parallel to the retinal surface and displaced in depth. Imaging was performed in three control subjects. RESULTS: A series of 8 depth-displaced en face images of retinal layers was reconstructed in each eye, which allowed enhanced visualization of the retinal structures and vasculatures. En face depth-displaced retinal images provided improved contrast compared with fundus images and delineated the foveal depression and the surrounding retinal vasculatures. CONCLUSIONS: An optical system for three-dimensional retinal imaging was developed that has potential as a tool for evaluation of retinal pathologies associated with chorioretinal diseases.

Nerve fiber layer thickness in glaucoma patients with asymmetric hemifield visual field loss.

PURPOSE: To investigate the presence of retinal nerve fiber layer (RNFL) thinning and determine the relationship between RNFL thickness and visual field sensitivity loss in glaucoma patients with asymmetric hemifield visual field loss. PATIENTS AND METHODS: Thirty glaucoma patients with asymmetric hemifield visual field loss and 30 normal control subjects were included in the study. RNFL thickness was measured by optical coherence tomography and visual field sensitivity was measured by automated perimetry. Glaucoma patients with advanced visual field loss restricted to 1 hemifield and early or absent glaucomatous field loss in the other hemifield on the basis of the visual field data were included. Visual field sensitivity and mean deviation (MD) were averaged separately in each of the 2 hemifields. The hemifields in each eye were categorized as early (MD>or=-6 dB) and advanced (MD<-6 dB) glaucomatous hemifields. RESULTS: RNFL thickness measurements in corresponding (eg, superior peripapillary quadrant with inferior hemifield) advanced glaucomatous hemifields (59+/-16 microm) were significantly (P<0.001) lower than in corresponding early glaucomatous hemifields (90+/-25 microm). The mean RNFL thickness in corresponding advanced and early glaucomatous hemifields were significantly lower than in normal control subjects (P<0.0001). On the basis of the normative database supplied by optical coherence tomography software, 100% and 43% of eyes had abnormal RNFL thickness in corresponding advanced and early glaucomatous hemifields, respectively. A linear correlation was found between RNFL thickness and MD in the early (r=0.6; P<0.001) and advanced (r=0.5; P=0.007) glaucomatous hemifields. CONCLUSIONS: RNFL thinning was present in corresponding hemifields of glaucomatous eyes with minimal visual field defect and correlated with visual field sensitivity loss. Measurement of RNFL thickness has potential for detection of early nerve fiber loss owing to glaucoma.

Quantitative thickness measurement of retinal layers imaged by optical coherence tomography.

PURPOSE: To report an image analysis algorithm that was developed to provide quantitative thickness measurement of retinal layers on optical coherence tomography (OCT) images. DESIGN: Prospective cross-sectional study. METHODS: Imaging was performed with an OCT3 commercial instrument in 10 visually normal healthy subjects. A dedicated software algorithm was developed to process the raw OCT images and detect the depth location of peaks from intensity profiles. Quantitative thickness measurements of three retinal layers, in addition to total retinal thickness, were derived. Total retinal thickness measurements obtained by the algorithm were compared with measurements provided by the standard OCT3 software. RESULTS: The total retinal thickness profile demonstrated foveal depression, corresponding to normal anatomy, with a thickness range of 160 to 291 microm. Retinal thickness measured by the algorithm and by the standard OCT3 software were highly correlated (R = 0.98). The inner retinal thickness profile predictably demonstrated a minimum thickness at the fovea, ranging between 58 to 217 microm along the 6-mm scan. The outer retinal thickness profile displayed a maximum thickness at the fovea, ranging between 66 to 107 microm along the 6-mm scan. The photoreceptor outer segment thickness profile was relatively constant along the 6-mm scan through the fovea, ranging between 42 to 50 microm. The intrasubject variabilities of the inner retina, outer retina, and photoreceptor outer segment thickness was 14, 10, and 6 microm, respectively. CONCLUSIONS: Thickness measurements of retinal layers derived from OCT images have potential value for objectively documenting disease-related retinal thickness abnormalities and monitoring progressive changes over time.

Inner and outer retinal thickness mapping of nonproliferative diabetic retinopathy by spectral-domain optical coherence tomography.

BACKGROUND AND OBJECTIVE: To report inner retinal thickness (IRT), outer retinal thickness (ORT), and total retinal thickness (TRT) mapping of nonproliferative diabetic retinopathy (DR). PATIENTS AND METHODS: Spectral-domain optical coherence tomography (SD-OCT) images were obtained in 31 study participants with nonproliferative DR. Semi-automated software generated IRT, ORT, and TRT maps. IRT, ORT, and TRT in each macular subfield were compared between groups with and without increased central subfield thickness. RESULTS: There were statistically significant differences in IRT, ORT, and TRT between groups (P < .007). In participants with nonproliferative DR with increased central subfield thickness, TRT was significantly increased in parafoveal and perifoveal inferior subfields (P < .001). In these subfields, both IRT and ORT were significantly increased (P < .007) compared to those in participants without increased central subfield thickness. CONCLUSION: Mapping of inner and outer retinal thickness shows promise for monitoring depth-specific thickness alterations by macular subfields due to DR.

Conjunctival microvascular haemodynamics in sickle cell retinopathy.

PURPOSE: To determine alterations in bulbar conjunctival microvascular haemodynamics in sickle cell retinopathy (SCR) subjects with focal macular thinning (FMT). METHODS: Conjunctival microcirculation imaging and spectral domain optical coherence tomography (SD-OCT) were performed in 22 subjects (eyes) diagnosed with SCR. Based on evaluation of SD-OCT retinal thickness maps, eyes were assigned to one of the two groups: with or without FMT. Conjunctival venular diameter and axial blood velocity were measured in multiple venules in each eye by customized image analysis algorithms. Measurements were then categorized into two vessel size groups (vessel size 1 and 2) and compared between FMT groups. A Pearson correlation coefficient was computed to assess the relationship between retinal thickness and axial blood velocity. RESULTS: Mean age, haematocrit, sickle cell haemoglobin type and median retinopathy score were not significantly different between the two groups (p ≥ 0.1). Retinal thickness in parafoveal and perifoveal temporal subfields was significantly lower in eyes with FMT as compared to eyes without FMT (p ≤ 0.04). There was a significant effect of FMT on axial blood velocity (p = 0.04), while the effect of vessel size was not significant (p = 0.4). In vessel size 1, axial blood velocity was lower in eyes with FMT than in eyes without FMT (p = 0.03), while in vessel size 2, there was no statistically significant difference between FMT groups (p = 0.1). In vessel size 1, there was a significant positive correlation between axial blood velocity and retinal thickness in the perifoveal (r = 0.48, p = 0.02) and parafoveal (r = 0.43, p = 0.04) temporal subfields. CONCLUSION: Conjunctival axial blood velocity in small venules is reduced in SCR subjects with focal macular thinning.

Feasibility of noninvasive imaging of chorioretinal oxygenation.

BACKGROUND AND OBJECTIVE: The feasibility of an optical system for noninvasive imaging of chorioretinal oxygenation was evaluated. Due to its depth discrimination, this optical section imaging technique has potential for differential imaging of oxygen tension in the chorioretinal vasculatures. MATERIALS AND METHODS: The method consisted of projecting a narrow laser line obliquely on the retina after intravenous injection of an oxygen-sensitive probe and imaging the phosphorescence emission. Due to the angle between the incident laser and imaging path, a phosphorescence optical section image of the retina was captured. The phosphorescence intensity was measured in the chorioretinal vasculatures. The method was tested in three rats while breathing 10% oxygen, 50% oxygen, and room air. RESULTS: On the phosphorescence optical section image, vasculatures appeared laterally displaced according to their depth location, displaying probe phosphorescence separately in the chorioretinal vasculatures. Oxygenation increased in all vasculatures with increased inhaled percent oxygen. Oxygenation in the retinal artery was 2.3, 1.9, and 1.6 times oxygenation in the retinal vein, capillary, and choroid, respectively. During hypoxia, oxygenation decreased by 28%, 18%, 22%, and 14% in the retinal vein, artery, capillary, and choroid, respectively. During hyperoxia, oxygenation increased by 30%, 45%, 36%, and 28% in the retinal vein, artery, capillary, and choroid, respectively. CONCLUSION: The results demonstrate the feasibility of this technique for noninvasive and separate imaging of chorioretinal oxygenation and its potential for three-dimensional oxygen tension imaging.

A Method for En Face OCT Imaging of Subretinal Fluid in Age-Related Macular Degeneration.

Purpose. The purpose of the study is to report a method for en face imaging of subretinal fluid (SRF) due to age-related macular degeneration (AMD) based on spectral domain optical coherence tomography (SDOCT). Methods. High density SDOCT imaging was performed at two visits in 4 subjects with neovascular AMD and one healthy subject. En face OCT images of a retinal layer anterior to the retinal pigment epithelium were generated. Validity, repeatability, and utility of the method were established. Results. En face OCT images generated by manual and automatic segmentation were nearly indistinguishable and displayed similar regions of SRF. En face OCT images displayed uniform intensities and similar retinal vascular patterns in a healthy subject, while the size and appearance of a hypopigmented fibrotic scar in an AMD subject were similar at 2 visits. In AMD subjects, dark regions on en face OCT images corresponded to reduced or absent light reflectance due to SRF. On en face OCT images, a decrease in SRF areas with treatment was demonstrated and this corresponded with a reduction in the central subfield retinal thickness. Conclusion. En face OCT imaging is a promising tool for visualization and monitoring of SRF area due to disease progression and treatment.

Feasibility of a method for en face imaging of photoreceptor cell integrity.

PURPOSE: To report a method for en face imaging of the photoreceptor inner and outer segment junction by spectral-domain optical coherence tomography (SD OCT) and to describe findings in normal subjects and patients with various retinal diseases. DESIGN: Observational case series. METHODS: SD OCT images were acquired from 6 normal subjects (mean age, 44 ± 11 years) and from 5 subjects with retinal diseases (mean age, 66 ± 22 years). A customized high-density SD OCT volume scan was acquired on the retina. SD OCT B-scan images were segmented automatically to extract intensity data along the inner and outer segment junction. Data obtained from the raster B-scans were combined to generate an inner and outer segment en face image in a 4.4 × 4.4-mm retinal area centered on the fovea. The foveal-to-parafoveal mean intensity ratio was measured, and repeatability was determined. An infrared scanning laser ophthalmoscope image was acquired and was cropped to provide a field of view similar to the inner and outer segment en face image. RESULTS: Inner and outer segment en face images generated in normal subjects provided clear visualization of the retinal vasculature, matching the vascular network observed in the infrared scanning laser ophthalmoscope image. In normal subjects, the foveal-to-parafoveal mean intensity ratio was 0.88 ± 0.06, and repeatability of measurements was, on average, 7%. In macular hole, a dark circular region was observed in the inner and outer segment en face image, indicative of photoreceptor cell loss. In age-related macular degeneration, the en face image displayed nonuniform texture corresponding to topographic variations in the inner and outer segment junction. In central serous retinopathy, areas of lower intensity were visible on the en face image corresponding to regions of prior neurosensory elevation. In cystoid macular edema, reduced intensity was present in the inner and outer segment en face image in areas with increased retinal thickness. In diabetic retinopathy, the inner and outer segment en face image displayed regions of reduced intensity resulting from edema, laser scars, or both. CONCLUSIONS: Detection of intensity abnormalities in the inner and outer segment en face image is useful for monitoring the integrity of photoreceptor cells in the course of disease progression and therapeutic intervention.

Thickness mapping of retinal layers by spectral-domain optical coherence tomography.

PURPOSE: To report normal baseline thickness maps for 6 retinal layers generated by segmentation of spectral-domain optical coherence tomography (SD-OCT) images in normal subjects. Intersubject thickness variability and thickness variations in 9 macular sectors were established. DESIGN: Prospective cross-sectional study. MATERIALS AND METHODS: SD-OCT imaging was performed in 15 normal subjects. Nineteen SD-OCT images were acquired, encompassing a 6 × 5-mm retinal area, centered on the fovea. Each image was analyzed using an automated segmentation algorithm to derive thickness profiles of 6 retinal layers. Thickness data obtained from all scans were combined to generate thickness maps of 6 retinal layers: nerve fiber layer, ganglion cell layer + inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer + photoreceptor inner segments, and photoreceptor outer segments. Mean and standard deviation of thickness measurements were calculated in 9 macular sectors and 6 retinal layers. Intersubject and intrasector thickness variations were established based on standard deviation of measurements. RESULTS: Minimum and maximum thickness of the nerve fiber layer were observed in the foveal and nasal perifoveal areas, respectively. The largest thickness variation among subjects and intrasector variability were observed in perifoveal areas. Thickness of the ganglion cell layer + inner plexiform layer and intersubject thickness variability were largest in parafoveal areas. The inner nuclear layer thickness was relatively constant in parafoveal and perifoveal areas and intrasector thickness variations were largest in the foveal area. The outer plexiform layer thickness was relatively constant in foveal and parafoveal areas and higher than in perifoveal areas. Intersubject thickness variability in inner nuclear layer and outer plexiform layer was relatively uniform in all macular sectors. The outer nuclear layer + photoreceptor inner segments thickness map displayed maximum thickness in the foveal area and intersubject thickness variability was largest superior to the fovea. Thickness of the photoreceptor outer segments layer, thickness variations among subjects, and intrasector thickness variability were relatively constant. There was a significant correlation between total retinal thickness derived by thickness mapping and SD-OCT commercial software. CONCLUSION: Normal thickness maps for 6 retinal layers were generated and thickness variations among subjects and macular areas were assessed. This technique is promising for investigating thickness changes attributable to disease in specific retinal layers and macular areas.

Differential macular morphology in patients with RPE65-, CEP290-, GUCY2D-, and AIPL1-related Leber congenital amaurosis.

PURPOSE: To evaluate genotypic and macular morphologic correlations in patients with RPE65-, CEP290-, GUCY2D-, or AIPL1-related Leber congenital amaurosis (LCA) using spectral-domain optical coherence tomography (SD-OCT). METHODS: SD-OCT macular scans were performed in 21 patients, including 10 with RPE65, 7 with CEP290, 3 with GUCY2D, and 1 with AIPL1 mutations. An image processing software was used to manually draw segmentation lines by three observers. Lamellar structure was evaluated based on the number of retinal layers on segmented images. Total retinal thickness was measured at the central macular and perifoveal areas by using an automated algorithm. RESULTS: All three patients with GUCY2D mutations (age range, 20-53 years) retained six retinal layers with visible photoreceptor inner/outer segment juncture (PSJ). However, the preservation of lamellar structures did not parallel better visual acuity. Patients with other mutations had poorly defined PSJ and disorganized retinal lamellar structures, where only one to three retinal layers could be observed. Patients with CEP290 mutations trended to have retention of the outer nuclear layer at the fovea and macular thickening, especially at younger ages. In patients with RPE65 (age range, 20-71 years) and AIPL1 mutations (age, 22 years), macular thickness was markedly decreased. Disorganization of retinal lamellar structures in the RPE65 group trended toward a worsening with increasing age. CONCLUSIONS: Variations of macular microstructures were observed among LCA patients with different genotypes. Disorganization of retinal lamellar structure was generally age related. Preservation of retinal microanatomic structures may not be associated with better visual acuity.