Anti-fumarase antibody promotes the dropout of photoreceptor inner and outer segments in diabetic macular oedema.

AIMS/HYPOTHESIS: In diabetic macular oedema (DMO), blood components passing through the disrupted blood-retinal barrier cause neuroinflammation, but the mechanism by which autoantibodies induce neuroglial dysfunction is unknown. The aim of this study was to identify a novel autoantibody and to evaluate its pathological effects on clinically relevant photoreceptor injuries. METHODS: Biochemical purification and subsequent peptide fingerprinting were applied to identify autoantigens. The titres of autoantibodies in DMO sera were quantified and their associations with clinical variables were evaluated. Two animal models (i.e. passive transfer of autoantibodies and active immunisation) were characterised with respect to autoimmune mechanisms underlying photoreceptor injuries. RESULTS: After screening serum IgG from individuals with DMO, fumarase, a Krebs cycle enzyme expressed in inner segments, was identified as an autoantigen. Serum levels of anti-fumarase IgG in participants with DMO were higher than those in diabetic participants without DMO (p < 0.001) and were related to photoreceptor damage and visual dysfunction. Passively transferred fumarase IgG from DMO sera in concert with complement impaired the function and structure of rodent photoreceptors. This was consistent with complement activation in the damaged photoreceptors of mice immunised with fumarase. Fumarase was recruited to the cell surface by complement and reacted to this autoantibody. Subsequently, combined administration of anti-fumarase antibody and complement elicited mitochondrial disruption and caspase-3 activation. CONCLUSIONS/INTERPRETATION: This study has identified anti-fumarase antibody as a serum biomarker and demonstrates that the generation of this autoantibody might be a pathological mechanism of autoimmune photoreceptor injuries in DMO.

Hyperreflective Foci in the Outer Retinal Layers as a Predictor of the Functional Efficacy of Ranibizumab for Diabetic Macular Edema.

Anti-VEGF drugs are as the first-line therapies for diabetic macular edema (DME). In this study, we investigated the association between hyperreflective foci in the outer retinal layers and functional efficacy in DME patients who received intravitreal ranibizumab (IVR) injections. We retrospectively reviewed 77 eyes of 71 patients with DME treated with pro re nata IVR injections for at least 12 months. We evaluated how baseline hyperreflective foci in the outer retinal layers on spectral domain optical coherence tomography images were associated with an improvement in logarithm of the minimum angle of resolution visual acuity (logMAR VA) at 12 months. Forty-three eyes with hyperreflective foci in the outer retinal layers had greater VA improvement than 34 eyes without such foci at 12 months. Multivariate analyses demonstrated that both logMAR VA and hyperreflective foci in the outer retinal layers at baseline were associated with VA improvement. Structural analyses revealed that the central subfield thickness was decreased and that the ellipsoid zone of photoreceptors was improved more significantly in eyes with hyperreflective foci in the outer layers than eyes without such lesions. Baseline hyperreflective foci in the outer retinal layers predict the functional efficacy of IVR injections for DME.

Disproportion of lamellar capillary non-perfusion in proliferative diabetic retinopathy on optical coherence tomography angiography.

PURPOSE: To characterise the non-perfused areas (NPAs) in the superficial and deep capillary layers (sNPAs and dNPAs, respectively) in the posterior pole in proliferative diabetic retinopathy (PDR) on wide-field optical coherence tomography angiography (OCTA) images. METHODS: We retrospectively reviewed 104 eyes of 70 patients with PDR from whom wide-field swept source OCTA images were acquired. sNPAs and dNPAs were manually measured in each quadrant of the inner (1-3 mm diameter), intermediate (3-6 mm), and outer (6-10 mm) rings centred on the fovea. Two qualitative findings, that is, segmented NPAs and periarteriolar NPAs, were also compared. RESULTS: The dNPAs were greater than the sNPAs (p<0.001) in each subfield. The outer ring had higher rates of deep NPAs than did the intermediate ring in the superior, inferior and temporal quadrants (p=0.010, p=0.004 and p<0.001, respectively), whereas no differences were detected in the nasal quadrant (p=1.000). Similarly, sNPA rates were higher in the outer ring than in the intermediate ring in the inferior and temporal subfields (p=0.003 and p<0.001, respectively). In 45 eyes with extensive NPAs, there were modest correlations between the dNPAs in the nasal and temporal quadrants in the intermediate (ρ=0.341, p=0.026) and outer (ρ=0324, p=0.032) rings, whereas sNPAs exhibited no associations. Segmented NPAs were delineated more frequently in the superficial layer than in the deep layer (p<0.001). Periarteriolar NPAs were more frequent in the deep layer (p<0.001). CONCLUSIONS: Three-dimensional assessment of wide-field OCTA promotes a better understanding of the enigmatic disproportion of lamellar NPAs in the posterior pole in PDR.

Predictor of Early Remission of Diabetic Macular Edema under As-Needed Intravitreal Ranibizumab.

The early remission of diabetic macular edema (DME) often occurs in eyes treated with anti-vascular endothelial growth factor (VEGF) treatment. We retrospectively reviewed and characterized eyes with early remission of DME at six months in 80 eyes under pro re nata (PRN) intravitreal ranibizumab (IVR) injections. The number of eyes without center-involved DME gradually increased and 14 and 20 eyes achieved remission of DME at 3 or 6 months, respectively, under the PRN regimen following three monthly loading doses. In particular, eyes with early remission at 6 months had smaller CSF thickness than those without the remission before and after the treatment except at the 1-month visit (P < 0.05); however, the changes in CSF thickness did not differ between them. VA and its changes were not different between eyes with and without remission. Multivariate analysis revealed that smaller CSF thickness at baseline predicted the early remission of DME under PRN IVR injections (odds ratio, 0.989; 95% confidence interval, 0.982-0.997; P = 0.008). These data elucidate the clinical characteristics of early remission of DME under PRN IVR injections and suggest that smaller CSF thickness at baseline is a novel predictor of early remission under PRN IVR injections for DME.

Anti-Fumarase Antibody as a Predictor of Functional Efficacy of Anti-VEGF Therapy for Diabetic Macular Edema.

Purpose: To evaluate whether baseline titers of anti-fumarase antibody are associated with visual prognosis after anti-VEGF treatment for diabetic macular edema (DME). Methods: In this retrospective study, we investigated 52 eyes of 52 DME patients who received intravitreal injections of anti-VEGF drugs (ranibizumab or aflibercept) after blood sampling at baseline. Optical coherence tomography (OCT) images were obtained at every monthly visit. The serum titer of anti-fumarase antibody at baseline was measured using ELISA. We evaluated the relationship between the titer of anti-fumarase antibody at baseline and visual acuity (VA) improvement at 12 months. Results: The serum titer of anti-fumarase IgG was related to the logMAR visual acuity (VA; R = 0.329, P = 0.017) and the disrupted ellipsoid zone (EZ; R = 0.364, P = 0.008) at baseline. The titer of this autoantibody was not associated with logMAR VA (R = -0.007, P = 0.980) but was associated with VA improvement (R = 0.465, P < 0.001) at 12 months upon anti-VEGF treatment. The transverse length of the disrupted EZ line was shortened at 12 months (P < 0.001), and restoration of the EZ line was correlated to the autoantibody titer (R = 0.396, P = 0.004) compared with the decrease in central subfield (CSF) thickness. Multivariate analysis showed that pretreatment logMAR VA (ß = 0.296, P = 0.045) and the autoantibody titer (ß = 0.328, P = 0.017) were associated with VA improvement after anti-VEGF treatment. In contrast, the titer was not associated with logMAR VA at 12 months. Conclusions: Anti-fumarase antibody is a novel serum biomarker predicting better functional efficacy of anti-VEGF treatment for DME.

Characteristics of Diabetic Capillary Nonperfusion in Macular and Extramacular White Spots on Optical Coherence Tomography Angiography.

Purpose: To compare the characteristics of macular and extramacular white spots on wide-field swept-source optical coherence tomography angiography (SS-OCTA) and optical coherence tomography (OCT) images in diabetic retinopathy (DR). Methods: We retrospectively reviewed 107 eyes of 64 patients with DR, of whom nominal 12 × 12 mm SS-OCTA images centered on the optic disc and ultrawide field photographs were acquired. White spots on fundus photographs corresponded to hyperreflective lesions in the superficial en-face OCT images, and the characteristics of these white spots were investigated. We compared such OCT findings with the vertical and horizontal extents of nonperfused areas (NPAs) on OCTA images. Results: We observed 136 white spots and corresponding hyperreflective lesions in 49 eyes. The hyperreflective lesions in the extramacular areas had greater areas (P < 0.001) and more frequently spanned from the nerve fiber layer to the outer plexiform layer (P < 0.001), while those in the macula were superficial. All of macular hyperreflective lesions were accompanied with nerve fiber layer defects, whereas only 18 (15.4%) of 117 extramacular lesions had them (P < 0.001). Comparative studies showed that most extramacular hyperreflective lesions corresponded to the NPAs in the whole layers on OCTA images, compared to the lamellar NPAs of the superficial layer in most of the macular lesions (P < 0.001). The NPAs extended to the peripheral side more frequently in the extramacular hyperreflective lesions compared with macular lesions (P < 0.001). Conclusions: This study proposed that most of the extramacular white spots may be discriminated from macular spots with respect to diabetic NPAs on OCTA images.

Anti-Hexokinase 1 Antibody as a Novel Serum Biomarker of a Subgroup of Diabetic Macular Edema.

Diabetic retinopathy (DR) induces the breakdown of the blood-retinal barrier and promotes neuroinflammation, although autoimmune responses to sequestered retinal antigens remain poorly understood. In this study, we investigated the autoantibodies for retinal antigens in sera from diabetic macular edema (DME) patients. Screening by immunoblotting demonstrated that IgG from 7 of 10 DME sera samples reacted to an ~102-kDa autoantigen from porcine retinas. Immunoprecipitation with autoantibodies from DME sera and subsequent mass spectrometry enabled us to identify hexokinase 1 as an autoantigen reactive to IgG from DME sera. IgG in 7 of 10 DME sera partially colocalized to hexokinase 1 in the outer plexiform layer of rodent retinas. Quantitative analyses using enzyme-linked immunosorbent assays revealed that the serum titers of this autoantibody were significantly higher in the DME sera than those in the sera from diabetic patients without DME, and 20 (24.1%) of the 83 DME serum samples had higher IgG titers than the cutoff value (mean + 2 standard deviations of the sera from diabetic patients without DR). Multivariate logistic regression analysis confirmed that the higher titer of anti-hexokinase 1 IgG was clinically feasible for the diagnosis of DME. These data identify anti-hexokinase 1 antibody as a serum biomarker of a subset of DME.

Association between characteristics of foveal cystoid spaces and short-term responsiveness to ranibizumab for diabetic macular edema.

PURPOSE: To investigate the association between the characteristics of foveal cystoid spaces and short-term responsiveness to ranibizumab treatment for diabetic macular edema (DME) at 3 months from the initial injection. METHODS: We retrospectively reviewed 66 eyes of 61 patients with center-involved DME who received three consecutive ranibizumab injections and following as-needed administrations. We evaluated the relationship between visual improvement at 3 months and the preoperative optical coherence tomography (OCT) parameters including hyperreflective foci, heterogeneous OCT reflectivity, mean levels of OCT reflectivity and height of foveal cystoid spaces. RESULTS: Twenty-three eyes without preoperative hyperreflective foci in the foveal cystoid spaces had significantly greater improvement in the logarithm of the minimum angle of resolution visual acuity (logMAR VA) at 3 months than 43 eyes with foci (P = 0.006). That was similar to the greater reduction in CSF thickness in eyes without lesions after treatment at the same time point (P < 0.001). VA improvement at 3 months was not associated with the height (R = 0.215, P = 0.083) or the reflectivity levels (R = -0.079, P = 0.538) of foveal cystoid spaces. There were no differences in VA changes between eyes with and without heterogeneous reflectivity in foveal cystoid spaces (P = 0.297). Multivariate analyses showed that logMAR VA and the absence of hyperreflective foci in foveal cystoid spaces were associated with VA improvement at 3 months. CONCLUSION: Hyperreflective foci in foveal cystoid spaces at baseline predict poorer short-term responsiveness to ranibizumab injections for DME.

Diabetic Nonperfused Areas in Macular and Extramacular Regions on Wide-Field Optical Coherence Tomography Angiography.

Purpose: To investigate the nonperfused areas (NPAs) in each subfield segmented by large arterioles on wide-field swept-source optical coherence tomography angiography (SS-OCTA) images in diabetic retinopathy. Methods: We retrospectively reviewed 101 consecutive eyes of 67 patients with severe nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), for whom 12 × 12-mm SS-OCTA images centered on the optic disc were acquired. Both eyes were included in 34 patients. NPAs in the whole retinal layers were measured in each subfield segmented by large arterioles encompassing both the superficial and deep layers. We compared the percentage of NPAs between individual subfields, considering the overlapping of the feeding arterioles. Results: Extramacular areas had higher rates of NPAs than macular areas in the inner (0.75-3 mm) and outer (3-5.5 mm) rings (P < 0.001 in both comparisons). The arteriolar arcades contacting the NPAs on the extramacular side were significantly longer than those contacting the NPAs on the macular side (P < 0.001). In particular, the extramacular areas between two arteriolar branches had a higher percentage of NPAs than those between two arterioles. The macular NPAs were greater in eyes with PDR than in those with severe NPDR, whereas there were no differences in the NPAs in the outer ring of extramacular areas. Conclusions: Wide-field OCTA images delineated that large arterioles residing in both the superficial and deep layers appear to be the perfusion boundaries, and the overlapping perfusion mediated via collateral vessels may affect the likelihood of diabetic NPAs in each subfield.

Decorrelation Signal of Diabetic Hyperreflective Foci on Optical Coherence Tomography Angiography.

Diabetic hyperreflective foci in the outer retinal layers are a clinically relevant finding on optical coherence tomography (OCT) images, although their characteristics remain to be elucidated. Here we investigated the decorrelation signal around hyperreflective foci on OCT angiography (OCTA) images in diabetic retinopathy (DR). We retrospectively reviewed sufficient quality OCTA images from 102 eyes of 66 patients that were obtained using split-spectrum amplitude-decorrelation angiography algorithm. Most confluent hyperreflective foci were randomly deposited or appeared in a radiating array on the en-face structural OCT images in the inner nuclear layer (INL) or Henle's fiber layer (HFL), respectively. Within the INL, hyperreflective foci were not accompanied by decorrelation signals and attached to capillaries on OCTA images. Decorrelation signals were sometimes delineated in hyperreflective foci in the HFL and other times appeared to be pseudopod-like or wrapping around hyperreflective foci, referred to as reflectance-decorrelated foci. The decorrelation signal intensity of hyperreflective foci in the HFL was associated with logMAR VA (R = 0.553, P < 0.001) and central subfield thickness (R = 0.408, P < 0.001) but not with DR severity. These data suggest that reflectance-decorrelated foci on OCTA images are clinically relevant as well as shed lights on the properties in diabetic hyperreflective foci.

Diabetic Neuroglial Changes in the Superficial and Deep Nonperfused Areas on Optical Coherence Tomography Angiography.

Purpose: To evaluate the relationship between lamellar capillary nonperfusion on optical coherence tomography angiography (OCTA) images and neuroglial changes on structural optical coherence tomography (OCT) images in diabetic retinopathy (DR). Methods: We retrospectively reviewed 101 consecutive eyes of 69 patients with DR. OCTA and OCT images within a 3 × 3-mm square centered on the fovea were acquired simultaneously. The nonperfused areas (NPAs) in the superficial capillary layer (sNPA) (from the nerve fiber layer [NFL] to the inner plexiform layer [IPL]), the deep layer (dNPA) (corresponding mainly to the inner nuclear layer [INL]), or both layers (bNPA) were measured individually along 10 transverse lines. The corresponding lamellar neuroglial changes also were evaluated on OCT images. Results: The transverse lengths of the sNPA, dNPA, and bNPA were 2.34% (interquartile range, 0.81-5.55), 0.61% (0-1.99), and 5.96% (4.02-10.88), respectively. The length of the sNPA was correlated significantly with the lengths of no boundary between the NFL and ganglion cell layer (GCL)/IPL and the spots with inverted OCT reflectivity in the sNPA. The transverse length of the dNPA was associated with the length of cystoid spaces in the INL or Henle's fiber layer (HFL) in the dNPA. There was a significant correlation between the transverse lengths of the bNPA and no boundary between the NFL and GCL/IPL within the bNPA. Conclusions: Systematic evaluation of the OCTA-OCT association showed structural changes in the neuroglial tissues corresponding to the lamellar NPAs and suggested the feasibility of layer-by-layer evaluation of the capillary nonperfusion in DR.

Clinical relevance of reduced decorrelation signals in the diabetic inner choroid on optical coherence tomography angiography.

Diabetes induces lesions of the retinal and choroidal capillaries, which promote the pathogenesis of diabetic retinopathy (DR). The decorrelation signals in optical coherence tomography angiography (OCTA) represent the blood flow and vascular structure, and three-dimensional OCTA images enable individual capillary layers to be evaluated separately. The current study documented that en-face OCTA images revealed spots of flow void in the choriocapillaris layer in eyes with DR. Quantitative investigation demonstrated that non-flow areas within the central subfield (CSF) increased in eyes with more severe DR grades. The non-flow areas in the choriocapillaris layer were also associated with poorer visual acuity (VA) in all 108 eyes. A modest correlation was noted between the areas of flow void and poorer VA in 69 eyes without DME, whereas the non-flow areas were not related to VA or to CSF thickness measured by OCT in 39 eyes with DME. In 12 eyes with ischemic maculopathy, the choriocapillaris layer beneath the disrupted ellipsoid zone of the photoreceptor (EZ) had greater areas of flow void than did the area beneath an intact EZ. These data suggested that disrupted choroidal circulation has clinical relevance and contributes to the pathogenesis of DR.

Relation between macular morphology and treatment frequency during twelve months with ranibizumab for diabetic macular edema.

PURPOSE: To investigate whether baseline optical coherence tomography (OCT) parameters can predict the treatment frequency of intravitreal ranibizumab (IVR) injections during the first year in patients with diabetic macular edema (DME) treated with pro re nata (PRN) IVR injections. METHODS: We retrospectively reviewed 68 eyes of 63 patients with center-involved DME who received IVR injections for 12 months or longer according to three monthly IVR injections followed by the PRN dosing. We measured the mean retinal thicknesses in the individual subfields of the Early Treatment Diabetic Retinopathy Study grid and evaluated the qualitative and quantitative parameters on OCT sectional images. We investigated the relationship between these OCT parameters at baseline and the number of IVR injections during the 12-month follow-up. RESULTS: Three loading doses were administered to 10 eyes; four to seven annualized IVR injections were administered to 34 eyes. The number of eyes that received IVR injections decreased gradually until month 6 and was almost constant from months 7 to 11. No relationships were seen between the treatment frequency and baseline systemic factors and the ophthalmic examination findings. Univariate analyses showed that the number of IVR injections during the first year was associated with the mean retinal thickness in the individual subfields and the transverse length of the disrupted external limiting membrane (ELM) and ellipsoid zone of the photoreceptors. Multivariate analysis showed a significant association with the thickness in the inferior subfield alone. The treatment frequency during the 12-month follow-up was not correlated with improved visual acuity but was associated with the decrease in the central subfield thickness and disrupted ELM. CONCLUSION: The retinal thickness in the inferior subfield predicts the treatment frequency during the first year in eyes with DME treated with PRN IVR injections.

Restoration of foveal photoreceptors after intravitreal ranibizumab injections for diabetic macular edema.

Anti-vascular endothelial growth factor drugs are the first-line treatment for diabetic macular edema (DME), although the mechanism of the visual acuity (VA) improvement remains largely unknown. The association between photoreceptor damage and visual impairment encouraged us to retrospectively investigate the changes in the foveal photoreceptors in the external limiting membrane (ELM) and ellipsoid zone (EZ) on spectral-domain optical coherence tomography (SD-OCT) images in 62 eyes with DME treated with intravitreal ranibizumab (IVR) injections. The transverse lengths of the disrupted EZ and ELM were shortened significantly (P < 0.001 and P = 0.044, respectively) at 12 months. The qualitative investigation also showed restoration of the EZ and ELM lines on SD-OCT images. The EZ at 12 months lengthened in 34 of 38 eyes with discontinuous EZ and was preserved in 16 of 21 eyes with complete EZ at baseline. VA improvement was positively correlated with shortening of the disrupted EZ at 12 months (ρ = 0.463, P < 0.001), whereas the decrease in central subfield thickness was associated with neither VA improvement nor changes in EZ status (ρ = 0.215, P = 0.093 and (ρ = 0.209, P = 0.103, respectively). These data suggested that photoreceptor restoration contributes to VA improvement after pro re nata treatment with IVR injections for DME independent of resolved retinal thickening.

Relationship between Functional and Structural Changes in Diabetic Vessels in Optical Coherence Tomography Angiography.

The decorrelation signals in optical coherence tomography angiography (OCTA) are derived from the flow of erythrocytes and concomitantly delineate the retinal vasculature. We compared the structural and functional characteristics of vascular lesions visualized in fluorescein angiography (FA), OCTA, and en-face OCT images in 53 eyes (28 patients) with diabetic retinopathy (DR). The foveal avascular zone (FAZ) areas in OCTA images in the superficial layer almost corresponded to those in FA images. The FAZ areas in the en-face OCT images in the superficial layer were smaller than those in the FA images and correlated with each other, which agreed with the finding that en-face OCT images often delineated the vascular structure in the nonperfused areas in FA images. Microaneurysms appeared as fusiform, saccular, or coiled capillaries in OCTA images and ringed, round, or oval hyperreflective lesions in en-face OCT images. OCTA and en-face OCT images detected 41.0 ± 16.1% and 40.1 ± 18.6%, respectively, of microaneurysms in FA images, although both depicted only 13.9 ± 16.4%. The number of microaneurysms in FA images was correlated with that in OCTA and en-face OCT images. Comparisons of these modalities showed the associations and dissociations between blood flow and vascular structures, which improves the understanding of the pathogenesis of DR.

Bilateral papillomacular retinoschisis and macular detachment accompanied by focal lamina cribrosa defect in glaucomatous eyes.

PURPOSE: We report here a patient with bilateral papillomacular retinoschisis with an enlarged glaucomatous optic nerve head cup and a focal lamina cribrosa defect, the findings of our clinical investigations of this case, and the chosen treatment and outcome. DESIGN: This is an observational case report. METHODS: Clinical examinations were performed using simultaneous confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT). The patient was treated by pars plana vitrectomy (PPV). RESULTS: The left eye had a macular detachment with a presumed acquired pit of the optic nerve, while the right eye did not have an obvious optic nerve pit. Enhanced depth imaging OCT showed focal lamina cribrosa defects in both eyes. PPV was performed on the left eye, which resulted in a re-attachment of the macula and improvement of the visual acuity. CONCLUSIONS: Our findings suggest that the pathological changes were most likely due to focal lamina cribrosa defects in both glaucomatous eyes. This type of maculopathy can be successfully treated with PPV.