Corneal Stroma Analysis and Related Ocular Manifestations in Recovered COVID-19 Patients.

Purpose: The purpose of this study was to assess the impact of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) on corneal stroma characteristics, ocular manifestations, and post-recovery refractive surgery outcomes after varying recovery durations. Methods: Fresh corneal lenticules from patients with post-coronavirus disease 2019 (COVID-19; recovered within 135 days) and healthy controls (HCs) after small incision lenticule extraction (SMILE) surgery were obtained for experimental validation of SARS-CoV-2 susceptibility, morphological changes, and immune response of the corneal stroma. Corneal optical density (CD) was measured using the Pentacam HR. Corneal epithelium thickness (ET) and endothelium parameters were evaluated by wide-field optical coherence tomography (OCT) and non-contact specular microscopy (SP-1P), respectively. All the patients were assessed after SMILE surgery until 3 month of follow-up. Results: The cornea was susceptible to SARS-CoV-2 with the presence of SARS-CoV-2 receptors (CD147 and ACE2) and spike protein remnants (4 out of 58) in post-recovery corneal lenticules. Moreover, SARS-CoV-2 infection triggered immune responses in the corneal stroma, with elevated IL-6 levels observed between 45 and 75 days post-recovery, which were then lower at around day 105. Concurrently, corneal mid-stromal nerve length and branching were initially higher in the 60D to 75D group and returned to control levels by day 135. A similar trend was observed in CD within zones 0 to 2 and 2 to 6 and in the hexagonal cells (HEX) ratio in endothelial cells, whereas ET remained consistent. Notably, these changes did not affect the efficacy, safety, or predictability of post-recovery SMILE surgery. Conclusions: SARS-CoV-2 induces temporal alterations in corneal stromal morphology and function post-recovery. These findings provided a theoretical basis for corneal health and refractive surgery management in the post-COVID-19 milieu.

Quantitative evaluation of morphological and functional changes in meibomian glands and lipid layer thickness in patients with and without keratoconus.

PURPOSE: To quantitatively evaluate the morphological parameters of meibomian glands (MGs) and lipid layer thickness (LLT) in patients with keratoconus (KC). METHODS: In this prospective, cross-sectional study, 164 eyes of 164 keratoconus patients and 64 eyes of 64 age-matched control subjects were included. An advanced automatic MG analyzer was used to quantitatively measure the morphological and functional parameters of MGs. Morphological and functional parameters of MGs, LLT, and other ocular surface parameters were compared between the control and KC groups. RESULTS: The mean meibomian gland diameter, length, square, and gland area ratio (GA) were all significantly decreased in the KC group (all P < 0.05), while no significant difference was observed in the gland tortuosity index (TI) and gland signal index (SI) between the KC and control groups (all P > 0.05). There was no significant difference in the number of total and incomplete blinking among patients with different stages of keratoconus (all P > 0.05). The gland diameter, square, and TI were all negatively associated with KC severity (all P < 0.05), while no significant difference was observed among all stages of KC in gland length, GA, and SI (all P > 0.05). Moreover, the LLTs were positively correlated with the gland diameter, square, GA, and TI and negatively correlated with anterior corneal curvature or KC severity (all P < 0.05). CONCLUSIONS: Atrophic morphological changes in the meibomian glands were closely correlated with the severity of keratoconus. Gland diameter may be a sensitive functional morphology metric of meibomian glands in patients with keratoconus.

Corneal Epithelial Remodeling Induced by Photorefractive Keratectomy Enhancement After Small-Incision Lenticule Extraction.

Purpose: To assess changes in corneal epithelial thickness (ET) within 9-mm diameter cornea after photorefractive keratectomy (PRK) retreatment after small-incision lenticule extraction (SMILE). Methods: A total of 28 eyes of 19 patients with mean spherical equivalent of -1.30 ± 0.60 D who underwent retreatment after SMILE were included in this retrospective study. ET mapped across a 9-mm diameter area was obtained using wide-field optical coherence tomography (OCT) before and at one, three, and six months after surgery. The ET changes were compared between the different time points and analyzed zones. Results: Before enhancement, the ET were 63.64 ± 6.01 µm and 61.25 ± 4.32 µm in central and paracentral zones, respectively. The ET of central and paracentral zones significantly decreased at one month and subsequently increased until six months. Six months after surgery, significant epithelial thickening occurred in 2- to 9-mm diameter cornea (all P < 0.05), whereas no significant change was observed in central 2-mm diameter cornea (P = 0.460). There was no significant difference in the ET between the central and paracentral zones (P = 1.00). The degree of myopic correction significantly correlated with the average ET in the central (P = 0.046) and paracentral (P = 0.033) zones at six months after PRK enhancement. No significant correlation was detected between the average ET of all zones and the postoperative spherical equivalent at six months after surgery (all P > 0.05). Conclusions: PRK enhancement did not alter the overall trend of corneal epithelial remodeling induced by SMILE. An asymmetric and flatter lenticule-like pattern of epithelial remodeling was observed six months after surgery, which did not affect the refractive outcomes. Translational Relevance: An asymmetric and centrally flattened lenticule-like pattern of epithelial remodeling was observed after PRK enhancement. Surgeons should consider expanding the intended optical zones for enhancement surgery after SMILE.

Ocular features in myopic eyes with longer horizontal ciliary sulcus diameters for intended implantable collamer lens surgery.

PURPOSE: To assess the ocular anterior segment characteristics in myopic eyes intended for ICL surgery with horizontal ciliary sulcus-to-sulcus (STS) diameters being greater than vertical STS diameters. METHODS: This retrospective, comparative case study included 1230 eyes of patients who underwent ICL implantation for the treatment of myopia or myopic astigmatism at the Zhongshan Ophthalmic Center from September 2020 to November 2021. The myopic eyes were divided into two groups according to the relatively long diameter of the ciliary sulcus. General parameters and anterior chamber parameters were compared between the two groups. RESULTS: 1230 eyes of 694 patients were included. The proportion of myopic eyes with longer horizontal STS diameters was 4.63%. Horizontal STS distances exceeding vertical meridians in these eyes were mainly attributed to the shortening of vertical STS distances (horizontal STS: P = 0.112; vertical STS: P < 0.001). Eyes with longer horizontal meridians of the ciliary sulcus displayed larger steep keratometry value (P = 0.001), larger corneal volume (P = 0.002), larger corneal astigmatism (P < 0.001), larger ocular residual astigmatism (P = 0.017), worse visual acuity (logMAR UDVA: P = 0.021; logMAR CDVA: P = 0.001), and more iridociliary cysts (P = 0.017) compared to eyes with vertically oval shapes. CONCLUSION: Myopic eyes with longer horizontal STS diameters are commonly accompanied by a change in corneal morphology and more iridociliary cysts. The anatomical features of the ciliary sulcus should be given sufficient consideration to ICL size and placement selection, contributing to more personalized and precise surgery.

Predicting post-operative vault and optimal implantable collamer lens size using machine learning based on various ophthalmic device combinations.

BACKGROUND: Implantable Collamer Lens (ICL) surgery has been proven to be a safe, effective, and predictable method for correcting myopia and myopic astigmatism. However, predicting the vault and ideal ICL size remains technically challenging. Despite the growing use of artificial intelligence (AI) in ophthalmology, no AI studies have provided available choices of different instruments and combinations for further vault and size predictions. This study aimed to fill this gap and predict post-operative vault and appropriate ICL size utilizing the comparison of numerous AI algorithms, stacking ensemble learning, and data from various ophthalmic devices and combinations. RESULTS: This retrospective and cross-sectional study included 1941 eyes of 1941 patients from Zhongshan Ophthalmic Center. For both vault prediction and ICL size selection, the combination containing Pentacam, Sirius, and UBM demonstrated the best results in test sets [R2 = 0.499 (95% CI 0.470-0.528), mean absolute error = 130.655 (95% CI 128.949-132.111), accuracy = 0.895 (95% CI 0.883-0.907), AUC = 0.928 (95% CI 0.916-0.941)]. Sulcus-to-sulcus (STS), a parameter from UBM, ranked among the top five significant contributors to both post-operative vault and optimal ICL size prediction, consistently outperforming white-to-white (WTW). Moreover, dual-device combinations or single-device parameters could also effectively predict vault and ideal ICL size, and excellent ICL selection prediction was achievable using only UBM parameters. CONCLUSIONS: Strategies based on multiple machine learning algorithms for different ophthalmic devices and combinations are applicable for vault predicting and ICL sizing, potentially improving the safety of the ICL implantation. Moreover, our findings emphasize the crucial role of UBM in the perioperative period of ICL surgery, as it provides key STS measurements that outperformed WTW measurements in predicting post-operative vault and optimal ICL size, highlighting its potential to enhance ICL implantation safety and accuracy.

Comparison of Refractive Outcomes and Optical Zone Decentration After SMILE Between Patients With Symmetrical and Asymmetrical High Astigmatism.

PURPOSE: To compare the refractive outcomes and optical zone decentration between patients with symmetrical and asymmetrical high astigmatism after small incision lenticule extraction (SMILE). METHODS: This was a prospective analysis of 89 patients (152 eyes) with myopia and astigmatism of more than 2.00 diopters (D) treated with the SMILE procedure. There were 69 eyes with asymmetrical topographies (asymmetrical astigmatism group) and 83 eyes with symmetrical topographies (symmetrical astigmatism group). Decentralization values were assessed using the tangential curvature difference map preoperatively and 6 months after surgery. Decentration, visual refractive outcomes, and induced changes in corneal wavefront aberrations were compared between the two groups 6 months postoperatively. RESULTS: Both groups achieved favorable visual and refractive outcomes, with a mean postoperative cylinder of -0.22 ± 0.23 and -0.20 ± 0.21 D in the asymmetrical and symmetrical astigmatism groups, respectively. In addition, visual and refractive outcomes and induced changes in corneal aberrations were comparable between the asymmetrical and symmetrical astigmatism groups (all P > .05). However, the total and vertical decentration in the asymmetrical astigmatism group was greater than that in the symmetrical astigmatism group (all P < .05), whereas no significant differences were found in the values of horizontal decentration between the two groups (P > .05). There was a weak positive correlation between induced total corneal higher order aberrations and total decentration (r = 0.267, P = .026) in the asymmetrical astigmatism group but not in the symmetrical astigmatism group (r = 0.210, P = .056). CONCLUSIONS: An asymmetrical corneal surface might affect treatment centration after SMILE. Subclinical decentration may be associated with the induction of total higher order aberrations, but it did not affect high astigmatic correction or induced corneal aberrations. [J Refract Surg. 2023;39(4):273-280.].

Lithium Chloride Exerts Anti-Inflammatory and Neuroprotective Effects by Inhibiting Microglial Activation in LPS-Induced Retinal Injury.

Purpose: To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. Methods: In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. Results: In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. Conclusions: This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Integrative transcriptomics analysis and experimental validation reveal immunomodulatory patterns in keratoconus.

Keratoconus is a progressive disorder of the cornea and is typically considered a noninflammatory disease. However, increasing evidence indicates that immune disorders play an essential role in keratoconus progression, but the immune-related etiology remains elusive. Here, we comprehensively utilized bioinformatics approaches and experimental methods to explore the potential immunoregulatory mechanism of keratoconus progression. Transcriptomics data containing two keratoconus patient groups was derived from the public dataset GSE151631. The intersection of genes and known immunological genes was used to obtain differentially expressed immune-related genes. We utilized various protein clustering algorithms to screen out and validated the hub immune-related genes, and further explored their potential biological functions via gene annotation and pathway enrichment analyses. Moreover, the underlying immune landscape and drug targets were predicted by immune cell infiltration analysis and drug-gene interaction analysis. Furthermore, keratoconus-related immunoregulatory competitive endogenous RNA networks were constructed and experimentally validated. After filtering and experimental validation, nine keratoconus-associated immune-related genes were credible. Infiltrated monocytes might play an essential role in the progression of keratoconus. Moreover, eleven intersecting drugs targeting four genes, CCR2, CCR5, F2RL1, and ADORA1, were considered as potential druggable molecular targets for keratoconus. Furthermore, in the competitive endogenous RNA network, we identified several lncRNAs and miRNAs as critical noncoding RNAs regulating the hub genes. Overall, our data indicated that the immunomodulatory patterns had undergone changes in the pathogenesis of keratoconus, which might facilitate the understanding of keratoconus-related immune processes and provide novel insights into developing new immunotherapies for keratoconus.

Identification of miR-671-5p and Its Related Pathways as General Mechanisms of Both Form-Deprivation and Lens-Induced Myopia in Mice.

Animal models have been indispensable in shaping the understanding of myopia mechanisms, with form-deprivation myopia (FDM) and lens-induced myopia (LIM) being the most utilized. Similar pathological outcomes suggest that these two models are under the control of shared mechanisms. miRNAs play an important role in pathological development. Herein, based on two miRNA datasets (GSE131831 and GSE84220), we aimed to reveal the general miRNA changes involved in myopia development. After a comparison of the differentially expressed miRNAs, miR-671-5p was identified as the common downregulated miRNA in the retina. miR-671-5p is highly conserved and related to 40.78% of the target genes of all downregulated miRNAs. Moreover, 584 target genes of miR-671-5p are related to myopia, from which we further identified 8 hub genes. Pathway analysis showed that these hub genes are enriched in visual learning and extra-nuclear estrogen signaling. Furthermore, two of the hub genes are also targeted by atropine, which strongly supports a key role of miR-671-5p in myopic development. Finally, Tead1 was identified as a possible upstream regulator of miR-671-5p in myopia development. Overall, our study identified the general regulatory role of miR-671-5p in myopia as well as its upstream and downstream mechanisms and provided novel treatment targets, which might inspire future studies.

Transcriptome Sequencing Reveals Tgf-ß-Mediated Noncoding RNA Regulatory Mechanisms Involved in DNA Damage in the 661W Photoreceptor Cell Line.

Transforming growth factor ß (Tgf-ß), a pleiotropic cytokine, can enhance DNA repair in various cells, including cancer cells and neurons. The noncoding regulatory system plays an important role in Tgf-ß-mediated biological activities, whereas few studies have explored its role in DNA damage and repair. In this study, we suggested that Tgf-ß improved while its inhibitor LSKL impaired DNA repair and cell viability in UV-irradiated 661W cells. Moreover, RNA-seq was carried out, and a total of 106 differentially expressed (DE)-mRNAs and 7 DE-lncRNAs were identified between UV/LSKL and UV/ctrl 661W cells. Gene ontology and Reactome analysis confirmed that the DE-mRNAs were enriched in multiple DNA damaged- and repair-related biological functions and pathways. We then constructed a ceRNA network that included 3 lncRNAs, 19 miRNAs, and 29 mRNAs with a bioinformatics prediction. Through RT-qPCR and further functional verification, 2 Tgf-ß-mediated ceRNA axes (Gm20559-miR-361-5p-Oas2/Gbp7) were further identified. Gm20559 knockout or miR-361-5p mimics markedly impaired DNA repair and cell viability in UV-irradiated 661W cells, which confirms the bioinformatics results. In summary, this study revealed that Tgf-ß could reduce DNA damage in 661W cells, provided a Tgf-ß-associated ceRNA network for DNA damage and repair, and suggested that the molecular signatures may be useful candidates as targets of treatment for photoreceptor pathology.

Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma.

Purpose: Retinoblastoma (RB) is the most common type of aggressive intraocular malignancy in children. The alteration of immunity during RB progression and invasion has not yet been well defined. This study investigated significantly altered immune-associated genes and cells related to RB invasion. Methods: The differentially expressed immune-related genes (IRGs) in noninvasive RB and invasive RB were identified by analysis of two microarray datasets (GSE97508 and GSE110811). Hub IRGs were further identified by real time PCR. The single-sample gene set enrichment analysis algorithm and Pearson correlation analysis were used to define immune cell infiltration and the relationships between hub IRGs and immune cells. Cell viability and migration were evaluated by CCK-8 and Transwell assays. A xenograft mouse model was used to verify the relationship between Src homology 3 (SH3) domain GRB2-like 2 (SH3GL2) expression and myeloid-derived suppressor cells (MDSCs). Results: Eight upregulated genes and six downregulated IRGs were identified in invasive RB. Seven IRGs were confirmed by real-time PCR. Moreover, the proportions of MDSCs were higher in invasive RB tissues than in noninvasive RB tissues. Furthermore, correlation analysis of altered immune genes and cells suggested that SH3GL2, Langerhans cell protein 1 (LCP1) and transmembrane immune signaling adaptor TYROBP have strong connections with MDSCs. Specifically, decreased SH3GL2 expression promoted the migration of RB cells in vitro, increased the tumor size and weight, and increased the numbers of MDSCs in the tumor and spleen in vivo. Conclusions: This study indicated that SH3GL2 and MDSCs play a critical role in RB progression and invasion and provide candidate targets for the treatment of RB.

CircRNA SCMH1 regulates the miR-200a-3p/ZEB1 signaling axis to promote diabetes-induced retinal epithelial-mesenchymal transition.

Diabetic retinopathy (DR) is one of the common systemic complications of diabetes. Epithelial-mesenchymal transition (EMT) is required for DR progression. Previous studies have explored that circular RNAs (circRNAs) are crucial for DR development. Herein, we focused on the biological functions of circSCMH1 in DR. RT-qPCR determined the expression of circSCMH1, miR-200a-3p and ZEB1. EMT-related proteins were measured by Western blot. Gene combinations were validated by RIP and dual luciferase reporter assays. CCK-8, EdU, TUNEL staining and Transwell analysis were used to assess the cellular function. FISH analysis assessed the localization of circSCMH1 and miR-200a-3p. HE staining was used to detect retinal structures in a mouse DR model. High-glucose (HG) significantly increased circSCMH1 expression in ARPE-19 cells. Additionally, circSCMH1 silencing repressed proliferation, migration, and EMT in HG cells. Mechanistically, circSCMH1 positively regulated ZEB1 expression via targeting miR-200a-3p. Furthermore, circSCMH1 was observed to induce HG cell growth and EMT by regulating the miR-200a-3p/ZEB1 axis. Finally, we verified that downregulation of circSCMH1 or ZEB1 alleviated EMT in the retina of diabetic mice. These findings have implications for new therapeutic targets for DR.

Comparison of fresh and preserved decellularized human corneal lenticules in femtosecond laser-assisted intrastromal lamellar keratoplasty.

Substantial evidence has demonstrated the application of fresh and decellularized human corneal lenticules from increasing myopic surgeries. Further preservation of decellularized corneal lenticules would extend its clinical application. However, whether fresh and preserved decellularized lenticules have the same effects in vivo, including refractive correction, remains unclear. Here, we made comprehensive comparisons between fresh human lenticules (FHLs) and preserved decellularized human lenticules (DHLs). Another group of decellularized lenticules was combined with crosslinking for potential keratoconus therapy. Optical transparency, biomechanical properties, and fibrillar ultrastructure were analyzed to evaluate the DHLs and crosslinked DHLs (cDHLs) in vitro. The DHLs retained high transparency and regular ultrastructure, with genetic materials mostly being eliminated. The strength of lenticules in the cDHL group was markedly increased by crosslinking. Moreover, after storage in glycerol for 3 months, the lenticules were reimplanted into rabbit corneal lamellar pockets assisted by a femtosecond laser. The rabbits were followed for another 3 months. There were no obvious rejective complications in any of the three groups. From 1 week to 3 months postoperatively, the host corneas of the FHL group remained highly transparent, while slight hazes were observed in the DHL group. However, the corneas of the cDHL group displayed opacity throughout the 3-month postoperative period. Furthermore, all the lenticules could effectively induce corneal steepening and refractive changes. Taken together, our data indicated that FHLs are ideal inlay products, whereas preserved DHLs could be an alternative for intrastromal lamellar keratoplasty. Our study provides new insights into the clinical application of human lenticule recycling. STATEMENT OF SIGNIFICANCE: Currently, substantial evidence has demonstrated the application of fresh and decellularized human corneal lenticules from increasing myopic surgeries. Further preservation of decellularized lenticules would extend its clinical application. However, whether fresh and preserved decellularized lenticules have the same effects in vivo, including refractive correction, remains unclear. Herein, we decellularized human lenticules with or without mechanically strengthened crosslinking. After storage in glycerol for 3 months, the lenticules were reimplanted into rabbit corneas. Comprehensive comparisons were performed among fresh human lenticules (FHLs), decellularized human lenticules (DHLs) and crosslinked DHLs. Our study indicated that FHLs are ideal inlay products, whereas preserved DHLs could be an alternative for intrastromal lamellar keratoplasty. Our study provides new insights into the clinical application of human lenticule recycling.

Early visual quality outcomes after small-incision lenticule extraction surgery for correcting high myopic astigmatism.

BACKGROUND: To evaluate early optical quality outcomes after small-incision lenticule extraction (SMILE) surgery for correcting high myopic astigmatism. METHODS: This retrospective study enrolled 55 eyes from 37 patients who had preoperative myopic astigmatism of ≥2.00 diopters (D) who had been treated with SMILE surgery. Preoperatively, the mean cylinder was - 2.41 ± 0.54 D (range, - 2.00 D to - 4.50 D). The preoperative and postoperative visual outcomes, refraction, and higher-order aberration (HOA) at 1 and 3 months were compared. Refractive astigmatism changes were analyzed by the Alpins vector method. RESULTS: Three months after SMILE surgery, the average cylinder was - 0.14 ± 0.31 D, and the average astigmatism vector was - 0.09 D × 6.34°. The angle of error (AofE) was limited to within ±10°, and the magnitude of error was limited to within ±1.0 D in all patients. The correction index (CI) was 0.98 ± 0.07, the index of success (IOS) was 0.08 ± 0.13, and the flattening index (FI) was 0.97 ± 0.07. Significant positive correlations were found between IOS and |AofE| (P = 0.000); negative correlations were found between FI and |AofE| (P = 0.000). The postoperative total HOA, spherical aberration, vertical coma aberration, and trefoil 30° were increased significantly compared with preoperative measurements, and the increase in HOA was closely related to preoperative astigmatism (P < 0.05). CONCLUSIONS: SMILE has preferable outcomes for correcting high myopic astigmatism. Axis rotation during the surgery might influence the undercorrection of astigmatism. The increase of HOA after surgery is related to preoperative astigmatism.