Purpose:To investigate the role of calcitonin gene-related peptide (CGRP) in corneal epithelial barrier dysfunction in dry eye disease (DED). Methods:Benzalkonium chloride (BAC)-induced murine dry eye models and hyperosmotic-stressed human corneal epithelial cells-transformed (HCE-T) were used. Corneal nerve density and CGRP expression were assessed via whole-mount staining and Western blot. CGRP receptor components, including CRLR, RAMP-1, and CRCP, were evaluated by quantitative real-time PCR and immunofluorescence. Mice received a topical CGRP solution (50 µM) for four days. Barrier function was analyzed using Oregon Green Dextran (OGD) staining and transepithelial electrical resistance (TEER). Tight junction proteins, inflammatory markers, and PKA/CREB/NF-κB signaling pathways were examined via immunofluorescence, qPCR, and Western blot. Results:BAC exposure caused significant corneal nerve damage (P < 0.05) and a reduction of CGRP levels (P < 0.001), accompanied by upregulation of CGRP receptor components (CRLR, RAMP-1, CRCP; P < 0.05). Exogenous CGRP treatment restored corneal epithelial barrier function, as indicated by decreased OGD staining intensity (P < 0.001) and improved TEER (P < 0.001), while maintaining the localization of tight junction proteins. Mechanistically, CGRP increased p-PKACα (T197)/PKACα and p-CREB (S133)/CREB (P < 0.01), while reducing p-p65 and IL-1β, IL-6, TNF-α, MMP-9 (P < 0.01). These effects were observed in both BAC-induced DED mice and in hyperosmotic-stressed human corneal epithelial cells, demonstrating that CGRP exerts dual mechanisms in barrier repair and anti-inflammatory protection. Conclusions:CGRP deficiency and receptor upregulation may be involved in the pathological process of DED. Exogenous CGRP ameliorates corneal barrier dysfunction, which is associated with modulation of the PKA/CREB and NF-κB pathways.

Dry eye, a common eye disease globally, poses significant challenges to clinical diagnosis and management due to its complex pathogenesis and high incidence rate. The development of artificial intelligence (AI) technology has provided new opportunities for the analysis and auxiliary diagnosis of dry eye imaging. This expert consensus focuses on the classification and annotation methods of dry eye imaging, in line with the application needs of AI technology. It summarizes the scope and tasks of research on the classification and annotation of dry eye imaging and provides detailed standards for the principles and methods of classification and annotation of major imaging modalities, including lipid layer of the tear film, tear meniscus height, tear film breakup time, corneal fluorescein staining, and meibomian gland images. It also clarifies the tools and processes for classification and annotation. The consensus proposes systematic quality control requirements, including annotation consistency assessment, multi-round review, and data cleaning methods. Finally, the consensus summarizes the current challenges and proposes targeted solutions. The launch of this consensus aims to provide high-quality data support for the development of AI in dry eye, enhance the application effects of AI in dry eye diagnosis, disease monitoring, and personalized treatment, and offer scientific references and technical support for clinical and research applications of AI in the field of dry eye.

Host-microbiome interplay during development governs the homeostasis of various bodily surfaces, however, postnatal colonization of the microbiome and its impact on the homeostasis of ocular surface is still unclear. Here, the changes of the conjunctival microbiome in C57BL/6 J mice were tracked in 1-week-old neonates through to 8-week-old adult mice. This disclosed that changes in the conjunctival microbiome correlate with age, especially at the 2-week and 3-week time points, which, respectively, are accompanied by eyelid-opening and weaning. Antigen presenting cells were also recruited to the conjunctival epithelium after eyelid-opening, whilst an inhibition of microbial colonization at 2-to-3 weeks of age led to a disruption of mucosal homeostasis and aggravated the development of allergic eye disease. This study improves our understanding of the development of the conjunctival microbiome in mice, and provides an indication that early microbial colonization is required for the establishment of mucosal ocular surface homeostasis, the perturbation of which leads to increased susceptibility to allergic eye disease. Colonization of conjunctival microbiota during early postnatal life in mice promotes ocular surface homeostasis, whereas microbiota disruption by topical antibiotic delays ocular mucosal development and aggravates allergic eye disease.

Purpose Clonal analysis is a feasible method to evaluate the status of stem/progenitor cells in epidermal or limbus investigations. This study aimed to evaluate the clonal growth potential of meibomian gland (MG) epithelial cells using clonal analysis. Methods Mouse and human MG tissues were isolated and cocultured with 3T3 feeder cells. Immunofluorescent staining of K14, K6a, and PPARγ on MG clones was applied. Holoclones, meroclones and paraclones were categorized based on clonal area. Triple staining and tile scans provided a comprehensive view of MG clone formation. MG ductal and acinar clones were cultured separately to compare stem/progenitor cell characteristics. We further evaluated an age-related MGD (ARMGD) mouse model along with two human MG samples of different ages using clonal analysis. Crystal violet staining was employed to assess clone formation efficiency (CFE). Results Both mouse and human MG epithelial cells formed clones on the feeder layers, which enlarged over time. The expression of K14, K6a, and PPARγ was decreased in differentiated clones during development. The CFE of holoclones and meroclones was approximately 1 ‰ in mouse MG clones and approximately 2.5 ‰ in holoclones and 5.6 ‰ in meroclones in human MG clones. The CFE of holoclones generated by ductal epithelial cells was significantly higher than did acinar clones. In the ARMGD mouse model and human samples, smaller clones, reduced CFE, and decreased K14+, K6a+, and PPARγ+ cells in MG clones were identified. Conclusions Clonal analysis effectively evaluates stem and progenitor cells in MGs, revealing deterioration in these cells under MGD conditions.

NLRP3 proteins mainly act as inflammasome core components in cytosol, but was sparsely recorded to translocate into nuclei in some conditions, such as in human simplex virus (HSV)-infected corneas, in SV40 T-Ag-immortalized human corneal epithelial cell (HCEC) line, or during differentiation of naïve T cells. This study was designed to define whether or how SV40 T-Ag transfection per se caused NLRP3 translocation. It was demonstrated that infection of primary human corneal epithelial cells with lentivirus coding for SV40 T-Ag induced NLRP3 proteins' translocation into nuclei. Pull-down of NLRP3-containing complexes in HCEC nuclear proteins followed by mass spectrometry revealed 285 nuclear proteins interacting with NLRP3 proteins. Clustering analysis of these proteins showed that "RNA binding", "Nucleocytoplasmic transport" and "Viral carcinogenic pathway" were among the enriched molecular function terms or KEGG pathways. Structural modeling showed significant but differential affinities between NLRP3 proteins and histone subunits. Systemic Evolution of Ligands by EXponential enrichment (SELEX) was utilized to define DNA motifs potentially bound by NLRP3 proteins in vitro, and in-depth analysis of SELEXed motifs confirmed that the genes harboring those motifs were significantly associated with transcription and RNA processing. This study demonstrated that during the process of SV40 T-Ag-mediated corneal cell immortalization, NLRP3 proteins translocated into nuclei and behaved like a transcription factor. Besides confirming NLRP3 proteins' novel functions in non-immune cells or tissues like cornea, these findings also shed light on the mechanisms of virus-mediated immortalization or viral induced carcinogenesis.

Purpose:To investigate the ocular surface changes during progress of the Sjögren's Syndrome (SS), using a previously described IL14α transgenic mice (IL14α TG) SS model. Methods:The ocular surface of IL14α TG and C57BL/6 wild-type (WT) female mice were evaluated at the age of six, nine, 12, 15, and 18 months. Slit lamp microscopy observation, Oregon green dextran staining, Schirmer test, and periodic-acid-Schiff staining were assessed. Immunohistochemistry, immunofluorescence, and associated gene expression analysis by qPCR and ELISA were performed in cornea, conjunctiva, and lacrimal grand at different ages of the mice. Masson's trichome staining was conducted on lacrimal gland cryosections. Results:Compared with C57BL/6 WT mice, IL14α TG mice showed corneal barrier function damage and losses in conjunctival goblet cell density starting at nine months, whereas decreases in tear secretion started at 18 months of age. Significant increases in CD4+ T cell infiltration in the conjunctiva of IL14α TG mice was first observed at 6 months. Higher expression levels of inflammatory cytokines IL-17A, IFN-γ, IL-1β, and TNF-α in the conjunctiva, whereas MUC5AC and MUC5B had lower expression levels at nine months in the IL14α TG mice. However, lacrimal gland function-associated gene expression levels mostly decreased in IL14α TG mice at 12 months of age. Conclusions:Ocular surface tissue changes were involved in SS-like dry eye in a time-dependent manner in IL14α TG mice, and conjunctival T-cell infiltration may contribute to ocular surface pathological changes in an early stage of SS-related dry eye.

Purpose: This study aimed to determine the role of peroxisome proliferator-activated receptor alpha (PPAR alpha) on corneal epithelial wound healing. Methods: Ten-week-old PPAR alpha knockout (PPAR alpha(-/-)) mice and wild-type (WT) C57BL/6 mice and ex vivo cultured human corneal epithelial cells were used to investigate the function of PPAR alpha on corneal epithelial wound healing. A two-millimeter diameter of the mice's central corneal epithelium was removed to induce corneal epithelial injury. The expression of PPAR alpha during corneal epithelial wound healing was analyzed using immunofluorescent staining and quantitative RT-PCR. Histological and immunostaining techniques were used to evaluate corneal morphology, cell proliferation, and inflammatory response in WT and PPAR alpha(-/-) mice. PPAR alpha agonist fenofibrate was used to determine its effect on corneal epithelial wound healing. Results: PPAR alpha expression was found to significantly increase during corneal epithelial repair. PPAR alpha(-/-) mice exhibited delayed corneal epithelial wound healing compared to WT mice. PPAR alpha(-/-) mice displayed altered proliferative responses and distinct patterns of inflammatory infiltrates. Administration of fenofibrate to WT mice resulted in accelerated corneal epithelial repair and increased PPAR alpha expression and cell proliferation. In vitro studies using human corneal epithelial cells further supported the impact of fenofibrate on promoting corneal epithelial cell wound healing. Conclusions: PPAR alpha is a regulator of corneal epithelial wound healing, and its absence leads to delayed repair processes in the corneal epithelium.

Meibomian gland dysfunction (MGD) is a chronic abnormality of the Meibomian glands (MGs) that is recognized as the leading cause of evaporative dry eye worldwide. Despite its prevalence, however, the pathophysiology of MGD remains elusive, and effective disease management continues to be a challenge. In the past 50 years, different models have been developed to illustrate the pathophysiological nature of MGD and the underlying disease mechanisms. An understanding of these models is crucial if researchers are to select an appropriate model to address specific questions related to MGD and to develop new treatments. Here, we summarize the various models of MGD, discuss their applications and limitations, and provide perspectives for future studies in the field.

Fuchs endothelial corneal dystrophy (FECD) stands as the most prevalent primary corneal endothelial dystrophy worldwide, posing a significant risk to corneal homeostasis and clarity. Corneal endothelial cells exhibit susceptibility to oxidative stress, suggesting a nuanced relationship between oxidant-antioxidant imbalance and FECD pathogenesis, irrespective of FECD genotype. Given the constrained availability of corneal transplants, exploration into non-surgical interventions becomes crucial. This encompasses traditional antioxidants, small molecule compounds, biologics, and diverse non-drug therapies, such as gene-related therapy, hydrogen therapy and near infrared light therapy. This review concentrates on elucidating the mechanisms behind oxidant-antioxidant imbalance and the evolution of strategies to restore oxidant-antioxidant balance in FECD. It provides a comprehensive overview of both conventional and emerging therapeutic approaches, offering valuable insights for the advancement of non-surgical treatment modalities. The findings herein might establish a robust foundation for future research and the therapeutic strategy of FECD.

BACKGROUND:The ocular surface and lacrimal gland have a frontline position in mucosal immunology. However, there have been few updates to the immune cell atlas of these tissues in recent years.PURPOSE:To map the immune cells in murine ocular surface tissues and lacrimal gland.METHODS:Central and peripheral corneas, conjunctiva, and lacrimal gland were dissociated into single cell suspensions, followed by flow cytometry. Discrepancy of immune cells between the central and peripheral corneas was compared. In the conjunctiva and lacrimal gland, myeloid cells were clustered by tSNE and FlowSOM based on the expression of F4/80, Ly6C, Ly6G, and MHC II. ILCs, type 1 immune cells, and type 3 immune cells were analyzed.RESULTS:The number of immune cells in peripheral corneas was about 16 folds of that in central corneas. B cells accounted for 8.74% of immune cells in murine peripheral corneas. In the conjunctiva and lacrimal gland, most myeloid cells tended out to be monocytes, macrophages, and classical dendritic cells (cDCs). ILC3 were 6.28% and 3.63% of ILCs in the conjunctiva and lacrimal gland, respectively. Th1, Tc1, and NK cells were predominant type 1 immune cells. γδ T17 cells and ILC3 outnumbered Th17 cells among type 3 T cells.CONCLUSION:B cells resident in murine corneas were reported for the first time. Additionally, we proposed a strategy of clustering myeloid cells to better understand their heterogeneity in the conjunctiva and lacrimal gland based on tSNE and FlowSOM. Furthermore, we identified the ILC3 in the conjunctiva and lacrimal gland for the first time. Compositions of type 1 and type 3 immune cells were summarized. Our study provides a fundamental reference and novel insights for ocular surface immune homeostasis and diseases.

The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models-a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples-along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.

Purpose:Accurate quantification measurement of tear meniscus is vital for the precise diagnosis of dry eye. In current clinical practice, the measurement of tear meniscus height (TMH) relies on doctors' manual operation. This study aims to propose a novel automatic artificial intelligence (AI) system to evaluate TMH.Methods:A total of 510 photographs obtained by the oculus camera were labeled. Three thousand and five hundred images were finally attained by data enhancement to train the neural network model parameters, and 60 were used to evaluate the model performance in segmenting the cornea and tear meniscus region. One hundred images were used to test generalization ability of the model. We modified a segmentation model of the cornea and the tear meniscus based on the UNet-like network. The output of the segmentation model is followed by a calculation module that calculates and reports the TMH.Results:Compared with ground truth (GT) manually labeled by clinicians, our modified model achieved a Dice Similarity Coefficient (DSC) and Intersection over union (Iou) of 0.99/0.98 in the corneal segmentation task and 0.92/0.86 for the detection of tear meniscus on the validation set, respectively. On the test set, the TMH automatically measured by our AI system strongly correlates with the results manually calculated by the ophthalmologists.Conclusions:We developed a fully automated and reliable AI system to obtain TMH. After large-scale clinical testing, our method could be used for dry eye screening in clinical practice.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.

Purpose To determine the effect of obstructive sleep apnea syndrome (OSA) on lacrimal gland function and its mechanism. Methods Male mice aged seven to eight weeks were housed in cages with cyclic intermittent hypoxia to mimic OSA, and the control group was kept in a normal environment. Slit-lamp observation, fluorescein staining, and corneal sensitivity detection are used to assess cornea changes. Tear secretion was detected by phenol red cotton thread, and the pathological changes of lacrimal gland were observed by hematoxylin and eosin staining, oil red O staining, cholesterol and triglyceride kits, immunofluorescence staining, immunohistochemical staining, real-time polymerase chain reaction, transmission electron microscopy, and Western blot. Results Studies revealed a decreased tear secretion, corneal epithelial defects and corneal hypersensitivity. Myoepithelial cell damage, abnormal lipid accumulation, reduced cell proliferation, increased apoptosis and inflammatory cell infiltration in the lacrimal gland were also seen. Hifα and NF-κB signaling pathways, moreover, were activated, while Pparα was downregulated, in the lacrimal glands of OSA mice. Fenofibrate treatment significantly alleviated pathological changes of the lacrimal gland induced by OSA. Conclusion OSA disturbs the Hifα/Pparα/NF-κB signaling axis, which affects lacrimal gland structure and function and induces dry eye.

Corneal neovascularization (CNV) is one of the common blinding factors worldwide and how to treat CNV effectively is a knotty problem in clinical practice. Although, photothermal therapy (PTT) has been widely investigated in CNV management, it is still limited by the effective delivery of photothermal agents. Hereby, we proposed and validated an in vivo assembly drug delivery strategy, which composed of vascular targeting molecule (RGD-Bis(DPA-Zn), DRUG-1) and ultra-small nanoparticles (AuPt-ICG, DRUG-2). DRUG-2, with PTT and photoacoustic imaging (PAI) properties, could be co-assembled with DRUG-1 to form larger DRUG-1@2 nanocluster, accompanied by performance gains. To treat CNV, DRUG-1 was topically administrated via eye drops; and trapped at the target by the in situ co-assembled with DRUG-1, during which the dual-PAI system was applied to trace drug enrichment; finally, a safe NIR irradiation strategy was implemented to realize efficient CNV elimination. Moreover, the DRUG-1/2-combined treatment demonstrated better biosecurity and broke new ground for CNV theranostics.