Conditional Deletion of Activating Rearranged During Transfection Receptor Tyrosine Kinase Leads to Impairment of Photoreceptor Ribbon Synapses and Disrupted Visual Function in Mice

Purpose: The rearranged during transfection (RET) receptor tyrosine kinase plays a key role in transducing signals related to cell growth and differentiation. Ret mutant mice show abnormal retinal activity and abnormal levels and morphology of bipolar cells, yet die on the 21st day after birth as a result of renal underdevelopment. To extend the observation period, we generated the Ret conditional knockout Chx10-Cre;C-Retlx/lx mouse model and analyzed the retinal function and morphological changes in mature and aging Chx10-Cre;C-Retlx/lx mice.Methods: Retina-specific depletion of Ret was achieved using mice with floxed alleles of the Ret gene with CHX10-driven Cre recombinase; floxed mice without Cre expression were used as controls. Retinal function was examined using electroretinography (ERG), and 2-, 4-, 12-, and 24-month-old mice were analyzed by hematoxylin staining and immunohistochemistry to evaluate retinal morphological alterations. The ultrastructure of photoreceptor synapses was evaluated using electron microscopy.Results: The results of the ERG testing showed that b-wave amplitudes were reduced in Chx10-Cre;C-Retlx/lx mice, whereas a-waves were not affected. A histopathological analysis revealed a thinner and disorganized outer plexiform layer at the ages of 12 and 24 months in Chx10-Cre;C-Retlx/lx mice. Moreover, the data provided by immunohistochemistry showed defects in the synapses of photoreceptor cells. This result was confirmed at the ultrastructural level, thus supporting the participation of Ret in the morphological changes of the synaptic ribbon.Conclusion: Our results provide evidence of the role of Ret in maintaining the function of the retina, which was essential for preserving the structure of the synaptic ribbon and supporting the integrity of the outer plexiform layer.

Characterization and staging of outer plexiform layer development in human retina and retinal organoids

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is critical for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells, and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of Bassoon to the OPL with each stage (p<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (p<0.0001). Overall, this study defines stages of human OPL development through mid-gestation and establishes HROs as a model system that recapitulates key aspects of human photoreceptor-bipolar cell synaptogenesis in vitro.

Smooth borders between inner nuclear layer and outer plexiform layer predict fewer macular edema recurrences in branch retinal vein occlusion

We hypothesized the smoothness of the border between the inner nuclear layer (INL) and outer plexiform layer (OPL) associates with the frequency of macular edema (ME) recurrences secondary to branch retinal vein occlusion (BRVO). Thirty-seven consecutive eyes with BRVO treated with anti-vascular endothelial growth factor (VEGF) injections at 1-year follow-up were included. We manually traced the border between the INL and OPL within the 1.5-mm vertical line from the fovea on optical coherence tomography (OCT) images at the initial visit. The jagged ratio (JR), the border length divided by the spline curve length, was calculated. We performed univariate and multivariate regression analyses, including JR, patient characteristics, number of cystoid spaces in the INL, INL area, and outer retina area. Multivariate regression analysis showed JR significantly correlates with the total number of anti-VEGF injections (P < 0.0001). Moreover, the mean JR was significantly lower in the nine eyes receiving two or fewer injections than in the 28 eyes receiving three or more injections (1.02 ± 0.01 vs. 1.13 ± 0.06, P < 0.0001). A smooth border between the INL and the OPL on OCT images at the initial visit may be a biomarker for fewer ME recurrences in eyes with BRVO.

Increased expression of Protein S in eyes with diabetic retinopathy and diabetic macular edema

Protein S (PS) is a multifunctional glycoprotein that ameliorates the detrimental effects of diabetes mellitus (DM). The aim of this study was to evaluate the distribution of PS in diabetic retinopathy (DR) and diabetic macular edema (DME). This was a study of 50 eyes with DM (37 with DME, 6 with proliferative DR, and 7 with no DR) and 19 eyes without DM. The level of PS was measured by enzyme immunoassay and was compared between eyes with or without DM, with or without DME, and with severe DME (≥ 350 μm) or mild DME (< 350 μm). We also performed immunohistopathologic evaluations of post-mortem eyes and the cystoid lesions excised during surgery. The aqueous free PS was significantly higher with DM (7.9 ± 1.2 ng/ml, P < 0.01) than without DM (6.1 ± 0.7). The aqueous free PS was significantly elevated with DME (8.2 ± 1.2, P < 0.05) compared to proliferative DR (7.0 ± 1.0) and no DR (7.0 ± 0.7). Eyes with severe DME had significantly higher aqueous free PS than mild DME (8.5 ± 1.3 vs. 7.7 ± 1.0, P < 0.05). Immunohistochemistry showed PS in the outer plexiform layer of the retina and cystoid lesion. The higher expression of PS with DR and DME suggests that PS is involved in their pathogenesis.

Outer Plexiform Layer Angle: A Prognostic Factor for Idiopathic Macular Pucker Surgery

Purpose. To investigate the efficacy of idiopathic macular pucker (epimacular membrane) surgery and to identify the possible prognostic factor. Methods. This was a retrospective study which enrolled 38 patients with idiopathic macular pucker who underwent 23-gauge pars plana vitrectomy (PPV) with indocyanine green-assisted peeling of epiretinal membrane (ERM) and internal limiting membrane (ILM). Visual outcomes were assessed at the baseline and during the follow-up including best-corrected visual acuity (BCVA) and metamorphopsia score, as well as outer plexiform layer (OPL) angle and central retinal thickness (CRT) using spectral-domain optical coherence tomography (SD-OCT). A comparison was made between patients with the prepeeling CRTs ≥500 μm and those <500 μm. A comparison was also made between patients with the prepeeling OPL angles ≥130° and those <130°. Based on the prepeeling parameters, the correlations between various prepeeling and postpeeling visual functions were analyzed. Results. Mean follow-up was 36.07 ± 4.62 months (range 1.30–96.70 months). BCVA was significantly improved from 0.26 ± 0.03 to 0.67 ± 0.04 p < 0.001 ; metamorphopsia score was significantly reduced from 1.42 ± 0.16 to 0.61 ± 0.08 p < 0.001 ; CRT was significantly decreased from 519.62 ± 13.41 μm to 385.37 ± 8.97 μm p < 0.001 . Greater prepeeling OPL angle (≥130°) was associated with significantly greater BCVA improvement (Snellen E/LogMAR: p = 0.01 / 0.03 ) and greater metamorphopsia reduction p = 0.03 , as compared to smaller OPL angle (<130°) with less BCVA improvement and less metamorphopsia reduction. However, the final BCVA improvement and metamorphopsia reduction relevant to the prepeeling smaller CRT (<500 μm) did not significantly differ from that relevant to the prepeeling greater CRT (≥500 μm; p > 0.05 ). Endophthalmitis, retinal tear, or retinal detachment was not observed after peeling. Conclusion. Indocyanine green-assisted ERM/ILM peeling combined with small gauge vitrectomy is associated with significant visual acuity improvement and metamorphopsia reduction in patients with idiopathic macular pucker. Greater prepeeling OPL angle rather than CRT might act as a useful prognostic factor in predicting better final visual functional outcomes.