Retinal Blood Velocity and Flow in Early Diabetes and Diabetic Retinopathy Using Adaptive Optics Scanning Laser Ophthalmoscopy.

Using adaptive optics scanning laser ophthalmoscopy (AOSLO), we measured retinal blood velocity and flow in healthy control eyes and eyes of diabetic patients with or without retinopathy. This cross-sectional study included 39 eyes of 30 patients with diabetes (DM) with mild non-proliferative diabetic retinopathy (NPDR) or without retinopathy (DM no DR) and 21 eyes of 17 healthy age-matched controls. Participants were imaged with a commercial optical coherence tomography angiography (OCTA) device (RTVue-XR Avanti) and AOSLO device (Apaeros Retinal Imaging System, Boston Micromachines). We analyzed AOSLO-based retinal blood velocity and flow, and OCTA-based vessel density of the superficial (SCP), deep retinal capillary plexus (DCP), and full retina. Retinal blood velocity was significantly higher in eyes with DM no DR and lower in NPDR across all vessel diameters compared to controls. Retinal blood flow was significantly higher in DM no DR and lower in NPDR in vessel diameters up to 60 µm compared to controls. When comparing flow outliers (low-flow DM no DR eyes and high-flow NPDR eyes), we found they had a significantly different retinal vessel density compared to the remaining eyes in the respective groups. Retinal blood velocity and flow is increased in eyes with DM no DR, while these parameters are decreased in eyes with mild NPDR compared to healthy age-matched controls. The similarity of OCTA vessel density among outliers in the two diabetic groups suggests an initial increase followed by progressive decline in blood flow and OCTA vessel density with progression to clinical retinopathy, which warrants further investigation.

Growth of hollow cell spheroids in microbead templated chambers.

Cells form hollow, spheroidal structures during the development of many tissues, including the ocular lens, inner ear, and many glands. Therefore, techniques for in vitro formation of hollow spheroids are valued for studying developmental and disease processes. Current in vitro methods require cells to self-organize into hollow morphologies; we explored an alternative strategy based on cell growth in predefined, spherical scaffolds. Our method uses sacrificial, gelatin microbeads to simultaneously template spherical chambers within a hydrogel and deliver cells into the chambers. We use mouse lens epithelial cells to demonstrate that cells can populate the internal surfaces of the chambers within a week to create numerous hollow spheroids. The platform supports manipulation of matrix mechanics, curvature, and biochemical composition to mimic in vivo microenvironments. It also provides a starting point for engineering organoids of tissues that develop from hollow spheroids.

Knock-in of Cx46 partially rescues fiber defects in lenses lacking Cx50.

PURPOSE: Connexins 46 (Cx46) and 50 (Cx50) support lens development and homeostasis. Knockout (KO) of Cx50, but not Cx46, causes defects in lens fiber organization, F-actin enrichment, gap junction (GJ) size, ball-and-socket (BS) maturation, and GJ-associated protein distributions. To further determine the unique roles of Cx50 and Cx46, we investigated whether these defects persisted in Cx46 knock-in (Ki) lenses. Ki mice had Cx46 knocked-in to their Cx50 loci, where it was expressed under endogenous Cx50 promoters. METHODS: Fiber cell morphology and the distribution of lens membrane/cytoskeleton proteins from wild-type (WT), Ki, and Cx50 KO mice were visualized by immunofluorescent labeling and confocal microscopy. RESULTS: Cx46 Ki partially rescued Cx50 KO lens fiber defects. Three-week-old Ki lens fibers had typical F-actin distributions but were nonuniformly sized and disorganized. The Cx-associated proteins zonula occludens-1 (ZO-1) and ß-dystroglycan (ßDys) no longer localized to the nuclei but remained absent from GJs. BS formed, but this occurred with lower than WT frequency. BS appeared with greater frequency in 8-week-old Ki lenses, but so did aberrant balloon-like structures similar to those in Cx50 KO lenses. Unexpectedly, 8-week-old Cx50 KO and Ki cortical lens fibers were no longer disorganized. CONCLUSIONS: Cx identity is important for some aspects of fiber development (organization, Cx association with ZO-1 and ßDys) but not others (F-actin enrichment). Either Cx supports BS maturation, but the sparsity of BS and presence of balloon-like structures in Ki lenses suggest that Cx50 is more capable of doing so. The partial rescue of BS structures may support the rapid growth of cortical fibers to the improved growth of Ki lenses compared to Cx50 KO lenses at young ages. Neither absence of Cx50 nor presence of Ki Cx46 affects cortical fiber cell organization by the age of 8 weeks.

Connexin 50 Regulates Surface Ball-and-Socket Structures and Fiber Cell Organization.

PURPOSE: The roles of gap junction protein connexin 50 (Cx50) encoded by Gja8, during lens development are not fully understood. Connexin 50 knockout (KO) lenses have decreased proliferation of epithelial cells and altered fiber cell denucleation. We further investigated the mechanism for cellular defects in Cx50 KO (Gja8-/-) lenses. METHODS: Fiber cell morphology and subcellular distribution of various lens membrane/cytoskeleton proteins from wild-type and Cx50 KO mice were visualized by immunofluorescent staining and confocal microscopy. RESULTS: We observed multiple morphological defects in the cortical fibers of Cx50 KO lenses, including abnormal fiber cell packing geometry, decreased F-actin enrichment at tricellular vertices, and disrupted ball-and-socket (BS) structures on the long sides of hexagonal fibers. Moreover, only small gap junction plaques consisting of Cx46 (α3 connexin) were detected in cortical fibers and the distributions of the BS-associated beta-dystroglycan and ZO-1 proteins were altered. CONCLUSIONS: Connexin 50 gap junctions are important for BS structure maturation and cortical fiber cell organization. Connexin 50-based gap junction plaques likely form structural domains with an array of membrane/cytoskeletal proteins to stabilize BS. Loss of Cx50-mediated coupling, BS disruption, and altered F-actin in Cx50 KO fibers, thereby contribute to the small lens and mild cataract phenotypes.