Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids.

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.

Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.

Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.

Tissue engineering RPE sheet derived from hiPSC-RPE cell spheroids supplemented with Y-27632 and RepSox.

BACKGROUND: Retinal pigment epithelium (RPE) cell therapy is a promising way to treat many retinal diseases. However, obtaining transplantable RPE cells is time-consuming and less effective. This study aimed to develop novel strategies for generating engineered RPE patches with physiological characteristics. RESULTS: Our findings revealed that RPE cells derived from human induced pluripotent stem cells (hiPSCs) successfully self-assembled into spheroids. The RPE spheroids treated with Y27632 and Repsox had increased expression of epithelial markers and RPE-specific genes, along with improved cell viability and barrier function. Transcriptome analysis indicated enhanced cell adhesion and extracellular matrix (ECM) organization in RPE spheroids. These RPE spheroids could be seeded and bioprinted on collagen vitrigel (CV) membranes to construct engineered RPE sheets. Circular RPE patches, obtained by trephining a specific section of the RPE sheet, exhibited abundant microvilli and pigment particles, as well as reduced proliferative capacity and enhanced maturation. CONCLUSIONS: Our study suggests that the supplementation of small molecules and 3D spheroid culture, as well as the bioprinting technique, can be effective methods to promote RPE cultivation and construct engineered RPE sheets, which may support future clinical RPE cell therapy and the development of RPE models for research applications.

Macular pigment optical density of hyperopic anisometropic amblyopic patients measured by fundus reflectometry.

Purpose: Hyperopic anisometropia is a major cause of amblyopia and may be associated with macular pigment optical density (MPOD) reduction. To explore whether the MPOD changes in hyperopic anisometropic amblyopia, we measured the MPOD using fundus reflectometry in eyes with hyperopic anisometropic amblyopia and normal vision. Methods: This was a cross-sectional study conducted from January 2017 to June 2017. Forty subjects (25 males and 15 females) between the ages of 6 and 10 years were recruited. The subjects' eyes were divided into two groups: amblyopic eyes (best-corrected visual acuity (BCVA) not more than 20/25 or BCVA of two eyes differing by two or more lines) and fellow eyes. All enrolled subjects underwent a comprehensive ophthalmic examination, including extraocular motility assessment, cover-uncover testing, and refractive error (noncycloplegic), BCVA, axial length (AL), macular foveal thickness (MFT) and MPOD (Visucam® 200, Carl Zeiss Meditec AG, Germany). Results: The MPOD of amblyopic and fellow eyes was 0.12 ± 0.03 log units and 0.13 ± 0.04 log units, respectively, with a significant difference (P = 0.026). The MFT of amblyopic and fellow eyes was 241.28 ± 13.95 and 237.13 ± 16.02 µm, respectively, revealing that the MFT was significantly higher in amblyopic eyes than in fellow eyes (P = 0.028). Conversely, there was no correlation between the MPOD and MFT in the two groups. Conclusions: This study is the first to report that the MPOD is decreased in hyperopic anisometropic amblyopia. In this study, no correlation between the MPOD and MFT was found. In the future, factors that induce a decrease in the MPOD in eyes with hyperopic anisometropic amblyopia should be explored in a large-sample study with follow-up observation.

Hydrogel eye drops as a non-invasive drug carrier for topical enhanced Adalimumab permeation and highly efficient uveitis treatment.

Uveitis is one of the most popular blind-causing eye diseases worldwide. Adalimumab (ADA) is used for the uveitis treatment through systemic or intravitreal injection at the expense of systemic side effects and increased medical risks. Although eye drops, a non-invasive topical treatment, could be a potential strategy to reduce side effects, it remains challenging to apply due to limited bioavailability mainly linked to poor retention time and permeation capacity for eye biological barriers. Here, we reported hydrogel eye drops composed of low-deacetylated chitosan and ß-glycerophosphate as an ADA carrier and tested its toxicity, tolerability, intraocular permeability, and efficacy of non-invasive treatment for uveitis. It's found the ADA-loaded hydrogel eye drops were more efficient than free ADA both in permeation rate and clinical efficacy for uveitis, Overall, this study provides a friendly non-invasive strategy to improve drug permeation rate and uveitis treatment efficacy, which may be valuable to clinically ophthalmic medication.

Microenvironment-Triggered Degradable Hydrogel for Imaging Diagnosis and Combined Treatment of Intraocular Choroidal Melanoma.

Human choroidal melanoma (HCM) is one of the most common primary intraocular tumors and easily provokes liver metastases owing to the lack of sensitive and noninvasive therapeutic methods. Concerning the imaging diagnostics and therapeutic predicaments for choroidal melanoma, we designed microenvironment-triggered degradable hydrogels (RENP-ICG@PNIPAM:Dox-FA) based on ultrasmall (<5 nm) rare-earth nanoparticles (RENPs) with enhanced NIR-II luminescence. The ultrasmall diameter can significantly enhance the NIR-II luminescence performance of RENPs. RENPs were encapsulated by a dual-response PNIPAM hydrogel, which could release drug by responding to heat energy and glutathione under the tumor microenvironment. The in vitro/in vivo NIR-II imaging detection and antitumor activity were also compared systematically after different treatment conditions on ocular choroidal melanoma-1 cells and tumor-bearing mice, respectively. Besides, the degradability of the hydrogel composites under physiological conditions could be conducive to enhance the photothermal-chemotherapeutic effect and alleviate long-term biological toxicity. Our work on the microenvironment-triggered hydrogels with enhanced NIR imaging and easy metabolism may provide a promising strategy for sensitive and noninvasive imaging and phototherapy in ocular tumors.

A Comparative Study of Total Corneal Power Using a Ray Tracing Method Obtained from 3 Different Scheimpflug Camera Devices.

PURPOSE: We sought to assess the agreement of ray-traced corneal power values by 3 Scheimpflug tomographers tp construct the corresponding arithmetic adjustment factor in comparison with an automated keratometer (IOLMaster) and a conventional Placido-based topographer (Allegro Topolyzer). DESIGN: Prospective reliability analysis. METHODS: A total of 74 eyes from 74 healthy subjects who underwent corneal power measurements using Pentacam, Sirius, Galilei, IOLMaster, and Allegro Topolyzer were included. Ray-traced corneal power values, such as total corneal refractive power (TCRP), mean pupil power (MPP), total corneal power (TCP), mean keratometry (Km), and simulated keratometry (SimK) were recorded respectively and analyzed using one-way analysis of variance (ANOVA) and Bland-Altman plots. RESULTS: Among the 3 ray-traced corneal power values, TCRP and MPP did not differ significantly (P = 0.81), whereas TCP presented a slightly significant larger value (P < 0.001). Compared to Km or SimK, corneal power measurements by the ray tracing method exhibited significantly lower values (P < 0.001). Bland-Altman plots disclosed that the 3 Scheimpflug tomographers showed similar 95% limits of agreement after arithmetic adjustment compared with Km (-0.40 to 0.40 D, -0.39 to 0.39 D, and -0.35 to 0.34 D) or SimK (-0.50 to 0.51 D, -0.43 to 0.42 D, and -0.46 to 0.46 D). CONCLUSIONS: Ray-traced corneal power values obtained using 3 Scheimpflug tomographers with default diameter settings were similar, indicating that they could be used interchangeably in daily clinical practice. The 3 Scheimpflug tomographers were satisfactory in agreement after arithmetical adjustment compared with conventional automated keratometer or Placido-based topographer.