Serum amyloid A aggravates endotoxin-induced ocular inflammation through the regulation of retinal microglial activation.

Serum amyloid A (SAA) are major acute-phase response proteins which actively participate in many inflammatory diseases. This study was designed to explore the function of SAA in acute ocular inflammation and the underlying mechanism. We found that SAA3 was upregulated in endotoxin-induced uveitis (EIU) mouse model, and it was primarily expressed in microglia. Recombinant SAA protein augmented intraocular inflammation in EIU, while the inhibition of Saa3 by siRNA effectively alleviated the inflammatory responses and rescued the retina from EIU-induced structural and functional damage. Further study showed that the recombinant SAA protein activated microglia, causing characteristic morphological changes and driving them further to pro-inflammatory status. The downregulation of Saa3 halted the amoeboid change of microglia, reduced the secretion of pro-inflammatory factors, and increased the expression of tissue-reparative genes. SAA3 also regulated the autophagic activity of microglial cells. Finally, we showed that the above effect of SAA on microglial cells was at least partially mediated through the expression and signaling of Toll-like receptor 4 (TLR4). Collectively, our study suggested that microglial cell-expressed SAA could be a potential target in treating acute ocular inflammation.

ANATOMIC OUTCOMES OF LENS-SPARING VITRECTOMY FOR STAGE 3 OR 4 FAMILIAL EXUDATIVE VITREORETINOPATHY.

PURPOSE: To report the anatomic outcomes and retinal structure changes from lens-sparing vitrectomy (LSV) for eyes with Stage 3 or 4 familial exudative vitreoretinopathy (FEVR). METHODS: Overall, 133 consecutive eyes of 119 patients with Stage 3 (51 eyes) or 4 (82 eyes) FEVR who underwent LSV between January 2012 and May 2023 were retrospectively reviewed. RESULTS: One hundred twenty-nine eyes (97.0%) achieved traction relief through one LSV operation. The extent of retinal detachment improved in 98 eyes (73.7%), remained stable in 32 eyes (24.1%), and progressed in three eyes (2.3%). At long-term follow-up, 39 (29.3%) and 60 (45.1%) eyes had completely or partially reattached retina, respectively. The median change of venular angle was 3.6° (95% CI, 3.5-10.5; P < 0.001) and -9.9° (95% CI, -15.8 to -4.6; P < 0.001) for temporal and nasal vessels, respectively. The mean disk-fovea distance was 0.3 papillary diameter shorter (95% CI, -0.4 to -0.2; P < 0.001), and the mean temporal venular arcade distance was 0.02 papillary diameter larger (95% CI, -0.16 to 0.21; P = 0.361). CONCLUSION: These results suggest that LSV can relieve vitreoretinal traction and reattach the retina in late-stage FEVR eyes. Improvements in temporal and nasal venular angle and disk-fovea distance reflect positive retinal structure changes for patients.

Investigating the Impact of Dimer Interface Mutations on Norrin's Secretion and Norrin/ß-Catenin Pathway Activation.

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/ß-catenin signaling pathway. Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/ß-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin. Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/ß-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport. Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/ß-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

Intra-Anterior Chamber Injection of Ranibizumab in Advanced Pediatric Vitreoretinal Diseases.

Importance: Anti-vascular endothelial growth factor (VEGF) treatment through intravitreal or subretinal administrations has been proven effective for VEGF-driven pediatric vitreoretinal diseases but are not feasible for advanced cases, such as shallow traction retinal detachments or peripheral circumferential retinal detachments which adhere to the lens. Intra-anterior chamber injection (IAcI) of anti-VEGF may be a viable alternative in such cases but needs evaluation. Objective: To investigate the effects and safety of IAcI of anti-VEGF to treat VEGF-driven pediatric vitreoretinal diseases. Design, Setting, and Participants: This was a retrospective observational case series study conducted at Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine in China. The study included 14 eyes of 13 children diagnosed with vitreoretinal disease exhibiting elevated vascular activity between January and August 2023. Intervention: IAcI with ranibizumab. Main Outcomes and Measures: Retinal vascular abnormalities, vitreous hemorrhage resolution, and complications 1 month and 3 months after injection. Results: Of 13 patients included in this study, 12 were male. The mean age was 4.6 years (range, 1 month to 9 years). Six patients were diagnosed with familial exudative vitreoretinopathy, 4 with morning glory syndrome, 1 with retinopathy of prematurity, and 2 with chronic retinal detachments of unknown causes. At 1-month postoperative follow-up, vascular activity had decreased in 14 of 14 eyes. At 3-month follow-up, vascular activity had resolved in 7 of 14 eyes, persisted in 6 of 14 eyes, and reactivated in 1 of 14 eyes. On final observation, no complications were reported. Conclusions and Relevance: These findings support the possibility of treatment using IAcI with ranibizumab to decrease retinal vascular abnormalities in familial exudative vitreoretinopathy or retinopathy of prematurity or related conditions, but further studies are needed to understand more precise benefits and risks. This approach might be considered in cases where intravitreal or subretinal injection are not feasible, recognizing the limitations of these findings and that longer-term outcomes still need to be monitored.

Vascular development analysis: a study for tertiary anti-vascular endothelial growth factor therapy after second reactivation of retinopathy of prematurity.

Purpose: To observe the vascular development results of tertiary anti-vascular endothelial growth factor (anti-VEGF) therapy following spontaneous second reactivation of retinopathy of prematurity (ROP). Methods: This retrospective study included 22 infants (42 eyes) with Type 1 or aggressive ROP (A-ROP) who received three anti-VEGF drug treatments for ROP from January 2018 to December 2022. The vascular growth, possible associated risk factors, and the retinal vascularization (DB/DF ratio) were assessed. Results: The mean follow-up was 17.6 months. After the 3rd intravitreal injection, seven eyes showed complete vascularization (Group 1), while the remaining 35 eyes demonstrated persistent avascular retina (PAR) (Group 2). In Group 2, 17 eyes maintained a stable state and were classified in the regression subgroup. The other 18 eyes developed a 3rd reactivation (reactivation subgroup) and were treated with laser photocoagulation (LPC).Birth weight (BW) was significantly lower in Group 2 than in Group 1 (p < 0.001). The decision tree analysis shows that only infants weighing more than 1,250 g (17.50%) had a chance to achieve complete retinal vascularization. The possibility of PAR was higher in patients with BW <1,250 g than ≥1,250 g (70.00% vs. 12.50%). In addition, most infants with BW ≥ 1,290 g and initial ROP disease in Zone I or posterior Zone II developed PAR. Conclusion: Tertiary IVR can successfully treat a second ROP reactivation and improve peripheral retinal vascularization. BW is the most significant factor related to complete retinal vascularization. Our decision tree model may be helpful in predicting the prognosis of anti-VEGF drugs in the event of a second ROP reactivation.

Identification of Novel FZD4 Mutations in Familial Exudative Vitreoretinopathy and Investigating the Pathogenic Mechanisms of FZD4 Mutations.

Purpose: The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4. Methods: Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/ß-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells. Results: All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/ß-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment. Conclusions: We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.

Generative adversarial networks synthetic optical coherence tomography images as an education tool for image diagnosis of macular diseases: a randomized trial.

Purpose: This study aimed to evaluate the effectiveness of generative adversarial networks (GANs) in creating synthetic OCT images as an educational tool for teaching image diagnosis of macular diseases to medical students and ophthalmic residents. Methods: In this randomized trial, 20 fifth-year medical students and 20 ophthalmic residents were enrolled and randomly assigned (1:1 allocation) into Group real OCT and Group GANs OCT. All participants had a pretest to assess their educational background, followed by a 30-min smartphone-based education program using GANs or real OCT images for macular disease recognition training. Two additional tests were scheduled: one 5 min after the training to assess short-term performance, and another 1 week later to assess long-term performance. Scores and time consumption were recorded and compared. After all the tests, participants completed an anonymous subjective questionnaire. Results: Group GANs OCT scores increased from 80.0 (46.0 to 85.5) to 92.0 (81.0 to 95.5) 5 min after training (p < 0.001) and 92.30 ± 5.36 1 week after training (p < 0.001). Similarly, Group real OCT scores increased from 66.00 ± 19.52 to 92.90 ± 5.71 (p < 0.001), respectively. When compared between two groups, no statistically significant difference was found in test scores, score improvements, or time consumption. After training, medical students had a significantly higher score improvement than residents (p < 0.001). Conclusion: The education tool using synthetic OCT images had a similar educational ability compared to that using real OCT images, which improved the interpretation ability of ophthalmic residents and medical students in both short-term and long-term performances. The smartphone-based educational tool could be widely promoted for educational applications.Clinical trial registration: https://www.chictr.org.cn, Chinese Clinical Trial Registry [No. ChiCTR 2100053195].