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Purpose: This study aimed to compare the expression of vitamin D receptor (VDR) on the ocular surface of patients with dry eye disease (DED) and controls without ophthalmological pathologies., Methods: Patients with DED without previous treatment were studied and compared with healthy subjects. Ocular Surface Disease Index assessement and ocular surface cytology were performed in all patients. The immunohistochemical expression of VDR was evaluated using fully automated immunohistochemistry. The evaluation involved multiplying the percentage of nuclear-labeled cells (0-100) by their intensity (0-3), resulting in a score ranging from 0 to 300 (VDR H-score). Squamous metaplasia was morphologically evaluated using liquid-phase cytology with Papanicolaou/periodic acid-Schiff staining using Nelson's grade (scale 0-3, higher grade to higher metaplastic change)., Results: Eighteen patients with DED without previous treatment and 10 healthy subjects were studied. Squamous metaplasia was observed in 74% of patients with DED, in contrast to 0% in the control group. In patients with DED, there was a lower expression of VDR than in the control group (VDR H-score: 11.2 ± 13.9 vs. 80.9 ± 56; P = 0.0001). Furthermore, an inverse correlation was observed between Nelson grade and VDR H-score ( P = 0.0001, rs = -0.71). No correlation was observed between Ocular Surface Disease Index and VDR H-score., Conclusions: This is the first study to evaluate the VDR in patients with DED. These patients presented with a lower expression of VDR than healthy subjects. No correlation was found with more severe symptoms. Patients with DED also had a higher frequency of squamous metaplasia., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Aims: Conjunctival epithelium lines the inside of the eyelids and covers the sclera, thus providing stability to the eye surface. Goblet cells in conjunctival epithelium (CjGCs) are well known for their mucin-secretion function, which wet and protect the ocular surface, but other aspects are still not well understood. To expand our understanding beyond their mucin-secreting function, we investigated CjGC-secreted extracellular vesicles (EVs) and lipid mediators therein., Materials and Methods: Using histamine-mediated allergic inflammation in human primary CjGCs (HCjGCs) as a disease model, we quantified using ELISA a proinflammatory mediator PGE2 and two specialized pro-resolving mediators (SPMs) LXA4 and RvD1 in EVs secreted during allergic inflammation., Key Findings: At 18 h post histamine stimulation, the amount of LXA4 and RvD1 in EVs was notably higher compared to those in unstimulated. Interestingly, this increase was only observed in female EVs but not in males. The mean fold increase of LXA4 and RvD1 in female EVs was 3.9 and 3.4, respectively, but it was only 0.9 and 1.0 in male EVs. Supplying docosahexaenoic acid (DHA, the source of RvD1 and other SPMs) to the culture medium during the allergic inflammation resulted in even higher mean fold increase of 5.3 and 6.9 for LXA4 and RvD1 in female EVs, respectively, but it was only 0.5 and 0.8 in male EVs., Significance: We conclude that HCjGCs show a clear sex difference in allergic response. Our results may also provide a new insight into the male predisposition to severe forms of allergic conjunctivitis and potential improvement in disease care in the clinic., Competing Interests: Declaration of competing interest This work is supported by National Institutes of Health (NIH) R01EY029789, EY029789T32EY007145, F32EY035529, P30EY003790; Korean-American Scientists and Engineers Association (KSEA) Young Investigator Grant (C.L)., (Copyright © 2024. Published by Elsevier Inc.)

Objective: The cross-sectional study was designed to evaluate the association of ocular surface inflammation with systemic conditions in patients with systemic lupus erythematosus (SLE)., Methods: The study enrolled 30 SLE patients and 30 controls. Ocular symptoms were evaluated using the Ocular Surface Disease Index (OSDI) questionnaire. Tear samples from all participants were collected for tear multi-cytokine and chemokine concentration analysis. All participants were assessed for dry eye disease (DED), including Schirmer I test, tear break-up time (TBUT), corneal fluorescein staining (CFS), meibomian gland secretion (MGS), lid-parallel conjunctival folds (LIPCOF), corneal clarity, and symblepharon. Besides, all participants were also examined for conjunctival impression cytology to measure the density of conjunctival goblet cells (CGCs). The peripheral blood indicators from SLE patients were also collected to measure the SLE-associated autoantibody specificities and systemic inflammatory indicators. Pearson and Spearman's analysis were uesd to examine the correlation between tear cytokines, CGCs, DED-related indicators, and systemic conditions., Results: The two groups were matched for age and gender in this study. 36.67% of eyes (11 in 30) of SLE patients and 13.33% of eyes (4 in 30) of controls were diagnosed with DED. OSDI scores, abnormal TBUT percentages, CFS percentages, and DED grading were all higher in SLE patients than in control group, while density of CGCs was lower. There were no significant differences in Schirmer I test, MGS, LIPCOF, corneal clarity, and symblepharon between SLE patients and controls. The levels of tear chemokine (C-X-C motif) ligand 11 (CXCL11) and cytokine interleukin-7 (IL-7) in patients with SLE were significantly higher than those in control group. Moreover, among SLE patients, the severity of DED and the level of tear chemokine CXCL11 were significantly positively correlated with SLE-associated autoantibody specificities., Conclusion: Dry eye and tear cytokines and chemokines-mediated ocular surface inflammation persist in SLE patients and are associated with systemic conditions. Therefore, it is necessary for patients with SLE to combine systemic and ocular assessments., Competing Interests: Declarations Ethics approval and consent to participate All subjects participated in the program voluntarily and signed informed consent forms. This study received approval from the clinical Research Ethics Committee of The Second Xiangya Hospital of Central South University (Ethics No.: Ocular Surface Observation in patients with systemic immune Diseases, LYF2021028). All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The Coronavirus disease 2019 (COVID-19) pandemic has led to a global health crisis, including ocular symptoms, primarily targeting the Angiotensin-Converting Enzyme 2 (ACE2) receptor. PM2.5 air pollution may increase infection risk by altering ACE2 expression. Silicon Dioxide (SiO 2 ) and carbon black (CB), major components of PM2.5 from sands and vehicle emissions, were studied for their effects on ACE2 and Transmembrane Protease Serine 2 (TMPRSS2) expression in corneal and conjunctival cells, and ocular tissues. Human corneal epithelial cells (HCECs) and conjunctival epithelial cells (HCjECs) were exposed to nanoparticles (NPs) for 24 hours, assessing viability via WST-8 assay. TNF-α, IL-6, and IL-1β levels in the medium were measured. An in vivo rat study administered NPs via eye drops, with Rose Bengal staining to evaluate damage. ACE2, TMPRSS2, and Angiotensin II (AngII) protein expressions were quantified by Western blot. ACE2 expression in HCjECs increased with NP exposure, while it decreased in HCECs. CB exposure increased TNF-α, IL-6, and IL-1β levels in HCECs. In vivo, corneal exposure to CB decreased ACE2 expression, whereas conjunctival exposure to SiO 2 increased ACE2 expression. These changes suggest that SiO2 exposure may increase the risk of COVID-19 through the ocular surface, while CB exposure may decrease it., (© 2024. The Author(s).)

The precise role and innate immunological mechanisms underlying lymphangiogenesis in pterygium remain unclear. This study aimed to investigate the presence of M1 and M2 macrophages and their correlation with pro-lymphangiogenic activation and lymphatic endothelial expression in human pterygium stromal tissues. We analyzed human pterygium and subject-matched normal conjunctival tissues for the expression of these factors and conducted in vitro experiments to assess interactions between macrophages and pterygium fibroblasts. Myeloid and M1 macrophage markers were upregulated in pterygium. M1 macrophages were associated with the upregulation of pro-lymphangiogenic vascular endothelial growth factor C (Vegfc) in pterygium tissues and induced inflammatory signals in pterygium fibroblasts. In contrast, lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) expression was associated with M2 markers but not with M1 markers. Notably, the clinical severity of pterygium was inversely correlated with the expression of the M2 marker Cd163. These findings suggest that M1 and M2 macrophages play distinct roles in the pathogenesis of pterygium, with M1 macrophages enhancing lymphangiogenic stimulation and inflammatory responses, while M2 macrophages are associated with Lyve1 expression and reduced severity of pterygium. Understanding these mechanisms may advance our current understanding of lymphatic biology in pterygium., Competing Interests: Declaration of competing interest No conflict of interest exists for any author., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Several studies have reported the protective effects of mesenchymal stem cell-derived exosomes (MSC-Exos) in reducing inflammation and decreasing conjunctival goblet cell (CGC) loss in dry eye disease. However, whether MSC-Exos provide anti-inflammatory profiles in macrophages, thus contributing to CGC protection, has remained elusive., Methods: Macrophages were incubated with PKH26-labeled periodontal ligament mesenchymal stem cell-derived exosomes (PDLSC-Exos) for 12 h, and uptake of PDLSC-Exos by macrophages was observed by a confocal fluorescence microscope. The mRNA expression of TNF-α, IL-10, and Arg1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of TNF-α and IL-10 were quantified using western blotting. Then, CGCs were exposed to different macrophage supernatants and qRT-PCR was used to detect the Muc5ac mRNA expression of CGCs in response to or absence of cholinergic stimulation. ELISA was used to determine the Muc5ac secretion of CGCs in response to cholinergic stimulation., Results: The uptake of PDLSC-Exos by M1 macrophages facilitates M2 macrophage polarization with the elevated expressions of IL-10 and Arg1. In macrophage supernatant-treated CGCs systems, PDLSC-Exo-treated M1 macrophage supernatant significantly enhanced the Muc5ac expression of CGCs in response to, or in the absence of, cholinergic stimulation, while the addition of PDLSC-Exos to the control macrophage supernatant did not generate a change in Muc5ac expression. Conversely, the addition of PDLSC-Exos to the diluted control macrophage supernatant induced a significant increase in Muc5ac expression., Conclusion: PDLSC-Exos could protect CGCs against M1 macrophage-mediated inflammation, and the protective effects of PDLSC-Exos are partly attributable to their effects on M1 macrophages.

Purpose: To compare the effect of the ocular antiseptic treatments 0.05% chlorhexidine, 5% povidone-iodine (PI) and 5% betadine on cell viability and mucin secretion of primary cultured human goblet cells (GCs)., Method: GC viability was analysed using lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Expression of mucin was visualised by immunohistochemical MUC5AC staining., Results: PI and betadine significantly reduced GC survival compared to the control (mean cell survival 23 ± 6% and 23 ± 7%, respectively, p < 0.05), whereas chlorhexidine did not significantly affect GC viability (mean cell survival: 78 ± 17%), as measured by the LDH assay. Similar results were obtained from the MTT assay, where PI and betadine caused a significant loss of GCs (mean cell survival: 26 ± 12% and 26 ± 13%, respectively, p < 0.05). Chlorhexidine did not significantly alter GC survival compared to the control (mean cell survival: 79 ± 8%). PI and betadine caused a dispersion of mucin secretion, which chlorhexidine did not., Conclusion: The most used antiseptic treatments, PI and betadine, applied prior to ocular surgery are significantly more cytotoxic to conjunctival GCs than chlorhexidine treatment., (© 2024 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Objectives: To investigate the clinical characteristics and the expression of mucin 5AC (MUC5AC) in in diabetic and non-diabetic subjects with or without dry eye disease., Methods: A total of 399 participants (796 eyes) aged 50-80 years were enrolled in this study. Participants were divided into 4 groups: non-diabetic without dry eye group (normal group), non-diabetic with dry eye group, diabetic without dry eye group and diabetic with dry eye group. Demographic information, fasting plasma glucose (FBG), and glycated haemoglobin A1C (HbA1C) data were collected. Additionally, ocular surface disease index (OSDI) questionnaire, signs of dry eye, tear osmolarity, and meibomian glands were evaluated. Tear MUC5AC expression and conjunctival goblet cells density (GCD) were tested., Results: Compared with non-diabetic with dry eye group, diabetic with dry eye group showed significantly lower tear film osmolarity (TFO), but higher corneal fluorescein and conjunctival lissamine green staining scores. In comparison with diabetic without dry eye group, diabetic with dry eye group showed significantly higher TFO, corneal fluorescein and conjunctival lissamine green staining scorers. The MUC5AC concentration and GCD of diabetic with dry eye group was significantly lower than those of the non-diabetic with dry eye group. Diabetic subjects with higher HbA1c levels (≥7.8%) showed higher TFO and shorter fluorescein tear break time., Conclusion: Diabetics with dry eye exhibited notably higher corneal fluorescein and conjunctival lissamine green staining scores. Conjunctival goblet cells and MUC5AC were significantly reduced in diabetics. Higher TFO was associated with the duration of diabetes and HbA1c levels., (© 2024. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

The narrow intersection between the cornea and conjunctiva, otherwise known as the limbus, is purported to harbor stem cells (SCs) that replenish the ocular surface epithelium throughout life. Damage to this site or depletion of its SCs can have dire consequences for eye health and vision. To date, various SC and keratin proteins have been used to identify the limbus, however, none could definitively mark its boundaries. Herein, we use the mouse as a model system to investigate whether structural and phenotypic features can be used to define the limbus and its boundaries with adjacent tissues. We demonstrate that differentially aligned blood and lymphatic vessels, intraepithelial nerves, and basal epithelial cellular and nuclei dimensions can be used as structural landmarks of the limbus. Identification of these features enabled approximation of the limbal expanse, which varied across distinct ocular surface quadrants, with the superior nasal and inferior temporal limbus being the widest and narrowest, respectively. Moreover, label-retaining SCs were unevenly distributed across the ocular circumference, with increased numbers in the superior temporal and inferior temporal moieties. These findings will heighten our current understanding of the SC niche, be beneficial for accurately predicting SC distribution to improve their isolation and devising efficacious cell therapies, and importantly, aid the ongoing search for novel SC markers., (© The Author(s) 2024. Published by Oxford University Press.)

Post-operative fibrosis of the filtering bleb limits the success of glaucoma filtration surgery (GFS). To minimise subconjunctival scarring following GFS, treatment with antimetabolites such as Mitomycin C (MMC) has become standard practice; however, their use is associated with considerable side effects. This study aimed to investigate the anti-scarring properties of 3',4'-dihydroxyflavonol (DiOHF). GFS was performed in New Zealand white rabbits who received eye drops of DiOHF three times daily and vehicle eye drops after surgery ( n = 5) or a single intraoperative treatment of MMC ( n = 5). Blebs were imaged immediately following surgery and on days 7, 15, 21, and 28 for clinical examination. On day 28, eyes were harvested to assess collagen deposition, expression of α-SMA, oxidative stress, angiogenesis, fibroblast activity, and inflammation in the conjunctiva/Tenon's layer. At 7 and 28 days post-GFS, MMC-treated blebs were more ischaemic than DiOHF- or vehicle-treated blebs. On day 28, DiOHF treatment significantly suppressed collagen accumulation, CD31 expression, Vimentin expression, and CD45 expression compared to the vehicle control. No difference was observed in 3-Nitrotyrosine or αSMA expression between treatment groups. Treatment with DiOHF reduced conjunctival scarring and angiogenesis in rabbits with GFS, which was comparable to MMC. DiOHF may be a safer and more effective wound-modulating agent than conventional antifibrotic therapy in GFS.

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: To figure out the roles of tear inflammatory cytokines in Ocular graft-versus-host disease (oGVHD) symptoms by analyzing tear cytokine levels and related factors., Methods: This prospective study involved 27 post-HSCT patients and 19 controls with dry eye disease. Analyses included tear cytokine (IL-6, IL-10, and TNF-α), ocular surface evaluation, and conjunctival impression cell examination. Tear cytokine levels were evaluated in three grades of corneal epithelial lesions. The study also analyzed the correlation between tear cytokine levels and ocular surface parameters. Tear cytokine levels were then used in a Receiver Operating Characteristic (ROC) curve and linear regression model to predict oGVHD related factors., Results: IL-6 has good diagnostic efficacy in oGVHD related dry eye. Elevated levels of tear IL-6 and TNF-α were observed in the group with severe corneal epithelial lesions. IL-6 levels were positively correlated with corneal fluorescein staining (CFS), eyelid margin hyperemia, conjunctival lesions, and meibum secretion. IL-6 showed excellent predictive ability with Area Under the Curve (AUC) values all greater than 0.70 ( p  < 0.05). IL-10 and TNF-α were negatively correlated with the meibomian gland proportion and conjunctival goblet cell (GC) density, while TNF-α was positively correlated with CFS and eyelid margin hyperemia., Conclusion: Dry eye symptoms related to ocular GVHD, can be partly diagnosed and assessed using various tear cytokine level detection methods.

Purpose: To explore the role of the proinflammatory cytokine, macrophage migration inhibitory factor (MIF), in a murine model of dry eye disease (DED)., Methods: The role of MIF on DED was determined using genetically MIF deficient mice and pharmacological inhibition of MIF. DED was induced with 0.5 mg of scopolamine via subcutaneous injection in wild type (WT) and mice lacking MIF (Mif -/- ), three times a day for 21 days. DED signs, tear volume, ferning pattern and cytology impression were evaluated. Also, eye tissues were collected to determine transcripts of key inflammatory mediators and histopathological damage. In a second set of experiments, we neutralized MIF with ISO-1, an isozaxiline-derivative MIF tautomerase activity-inhibiting small molecule in WT mice, following an acute DED model for 10 days. ISO-1 was given starting on day 3 after DED induction and signs were evaluated, including a recovery phase in both experimental approaches., Results: When compared to WT, Mif -/- mice showed attenuated signs of DED like preserved mucin pattern and increased tear volume. Also, Mif -/- mice maintained conjunctival epithelial cells and less corneal damage, associated with lower levels of TNFα and IL-1β. At recovery phase, Mif -/- mice presented improved signs. Interestingly, in cornea and conjunctiva the absence of MIF selectively downregulated the transcription of inflammatory enzymes like inos and nox4 whereas displayed enhanced transcripts of il-4, il-13, tgfβ and cox2. Finally, pharmacological inhibition of MIF using ISO-1, replicated the above findings in the mouse model., Conclusion: MIF is a central positive mediator of the inflammatory process in experimental DED, thus, targeting MIF could be used as a novel therapy in ocular surface inflammatory pathologies.

Purpose: The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen., Methods: The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen., Results: Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1 , SLC35B4 , STEAP2 , ATP2B2 , NMNAT3 , and AKAP12 ., Conclusions: This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The protein epidermal growth factor (EGF), which plays a crucial role in promoting cell proliferation and survival, has recently demonstrated potential in reducing inflammation. In this study, we examined the impact of EGF on the anti-inflammatory and anti-proliferative properties of pterygium, a prevalent hypervascular proliferative disease affecting the ocular surface. In surgically excised tissues, markers for fibrotic and inflammatory signals, including VIM, ACTA2, FAP, MMP2, VCAM1, ICAM1, CD86, IL6, and IL1B were upregulated in the pterygium body stroma compared to the normal conjunctival stroma. EGF exerted anti-inflammatory and anti-vasculogenic effects on pterygial fibroblasts when co-cultured with M1 macrophages. Moreover, exosomes derived from EGF-preconditioned M1 macrophages suppressed the heightened inflammatory and vasculogenic signals in pterygial fibroblasts induced by exosomes from M1 macrophages. Paradoxically, the proliferation of pterygial fibroblasts was inhibited by EGF in the in vitro microenvironment with M1 macrophages, despite EGF being known as a growth factor. EGF-preconditioning of M1 macrophages rescued the increased proliferation of pterygial fibroblasts induced by exosomes from M1 macrophages. In conclusion, our findings demonstrate that EGF effectively mitigates inflammation and proliferation in pterygial fibroblasts within a microenvironment containing M1 macrophages., (© 2024. The Author(s).)

Purpose: Pterygium is an ocular surface disease characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva to the cornea and is associated with abnormal tear function caused by changes in tear composition and osmolarity. In this study, the effect of two different surgical techniques to remove primary pterygium: conjunctival autograft surgery (CAG) and amniotic membrane transplantation (AMT), on changes in MUC2 and MUC5AC tear mucins concentration were evaluated., Methods: Forty-four patients (>18 years old) with primary unilateral pterygium (> 1.0 mm long, measured from the limbus to the apex on the cornea) were randomly enrolled, and assigned to the AMT or CAG group by using the permuted block technique. Patients with systemic inflammatory diseases or other eye comorbidities were excluded from the study. Tear break-up time (TBUT) and best-corrected visual acuity (BCVA) assessments were performed before surgery and at 1, 3, and 6 months after surgery. Tears were collected concurrently with the clinical evaluations, and MUC2 and MUC5AC concentrations were subsequently measured by means of ELISA., Results: At 6 months after CAG or AMT, TBUT and BCVA were significantly lower (P < 0.05) in comparison with the baseline values in the study subjects. The tear mucin concentrations of both MUC2 and MUC5AC were significantly higher (P < 0.0001) in patients with pterygium before any surgical procedure than in healthy individuals. The concentration of MUC2 increased at 1 and 3 months after CAG surgery and decreased at 6 months; however, the MUC2 concentration decreased on the AMT group in all time point measurements. Interestingly, the MUC5AC concentration significantly increased at 1 month after AMT or CAG and then decreased at 3 and 6 months after surgery. Finally, an inverse correlation was found between both MUC2 and MUC5AC tear mucins concentration and the TBUT., Conclusions: These results suggest that pterygium excision via both CAG or AMT changes the concentrations of the tear mucins MUC2 and MUC5AC during the evaluated times, and these changes could affect tear film stability and clinical recovery after pterygium treatment., Translational Relevance: The tear film stability during pterygium excision was evaluated to determine adequate treatments.

Purpose: This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group., Methods: This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions., Results: Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of IL-5 and IL-6 in the corneal epithelium was higher in the KC group than in the control group, with IL-5 showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group., Conclusions: In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively., (Copyright © 2024 Molecular Vision.)

Purpose: To reveal the penetration of epinastine, an anti-allergic ophthalmic agent, into the eyelid and its distribution to the conjunctiva after administration of a cream formulation on rabbit eyelid skin., Study Design: Experimental study., Methods: Rabbits were treated with 0.5% epinastine cream on hair-shaved eyelids, followed by preparation of eyelid tissue slices to determine spatial tissue distribution of epinastine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification using laser-microdissected tissues and desorption electrospray ionization mass spectrometry imaging (DESI-MSI). In addition, following either eyelid application of 0.5% epinastine cream or ocular instillation of 0.1% epinastine eye drops, concentration-time profiles of epinastine in the palpebral conjunctiva and bulbar conjunctiva were determined using LC-MS/MS., Results: Laser microdissection coupled with LC-MS/MS analysis detected high concentrations of epinastine around the outermost layer of the eyelid at 0.5 h post-administration that gradually diffused deeper into the eyelid and was distributed in the conjunctival layer at 8 and 24 h post-administration. Similar time-dependent drug distribution was observed in high-spatial-resolution images obtained using DESI-MSI. Epinastine concentrations in the conjunctival tissues peaked at 4-8 h after administration of 0.5% epinastine cream and then decreased slowly over 72 h post-administration. In contrast, epinastine concentrations peaked quickly and decreased sharply after epinastine eye drop administration., Conclusion: After the application of epinastine cream to the eyelid skin, epinastine gradually permeated the eyelid. The compound was retained in the conjunctiva for 8-24 h post-administration, indicating that epinastine cream is a promising long-acting formulation for treating allergic conjunctivitis., (© 2024. The Author(s).)

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca 2+ i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca 2+ ] i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1 ) preincubation with the Ca 2+ chelator EGTA, 2 ) preincubation with the SERCA inhibitor thapsigargin (10 -6 M), which depletes ER Ca 2+ stores, 3 ) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4 ) preincubation with the voltage-gated calcium channel antagonist nifedipine (10 -5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10 -5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca 2+ ] i was significantly lower after chelating Ca 2+ i with 2 mM EGTA in Ca 2+ -free buffer. The peak [Ca 2+ ] i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10 -5 M and BzATP at 10 -4 M. Incubating CGCs with dantrolene alone decreased [Ca 2+ ] i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca 2+ i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca 2+ ] i . This study contributes to the understanding of diseases arising from defective Ca 2+ signaling in nonexcitable cells. NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca 2+ ] i ) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca 2+ ] i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca 2+ ] i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca 2+ ] i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca 2+ i homeostasis.

Purpose: To evaluate the therapeutic efficacy of topical application of a neurokinin-1 receptor (NK1R) antagonist in a rabbit model of nonallergic ocular redness. Methods: Nonallergic ocular redness was induced in rabbits by a single, topical application of dapiparzole hydrochloride eye drops (0.5%, 1%, 2%, or 5%). The NK1R antagonist L-703,606 was topically applied to the eye at the same time of induction or 20 min after induction, and phosphate buffered saline (PBS) treatment served as the control. Superior bulbar conjunctival images were taken every 30 s for the first 2 min, followed by every 4 min for 8 min, and then every 10 min until 1 h. The severity of ocular redness was evaluated on the images using ImageJ-based ocular redness index (ORI) calculations. Results: The ORI scores were significantly increased after the application of 0.5%, 1%, 2%, or 5% dapiparzole at each time point evaluated, with the most severe redness induced by the 5% dapiprazole that led to a maximal mean increase in ORI score of 14 at 20 min post-induction and thus used for subsequent evaluation of therapeutic efficacy of NK1R antagonism. Topical L-703,606, when applied at the same time as dapiprazole induction, significantly suppressed the increase of ORI scores at all time points (∼40% decrease). Furthermore, when applied at 20 min after dapiprazole induction, L-703,606 rapidly and effectively suppressed the increase of ORI scores at 30, 40, 50, and 60 min (∼30% decrease). Conclusions: Topical blockade of NK1R effectively prevents and alleviates nonallergic ocular redness in a novel animal model.

Objective: This study aims to investigate the effect of NADPH oxidase 4 (NOX4)-mediated inflammation on concanavalin A (ConA)-induced dry eye syndrome (DES) in mice. Methods: Thirty-six mice were randomly divided into Control, Model, no-load Control, and NOX4 interference group. Adenovirus was injected (10 μL) into the lacrimal glands of both eyes of mice in no-load Control group and NOX4 interference group. Four days after adenovirus injection, the Control group was injected with phosphate-buffered saline, and the other groups were injected with ConA (200 μg) in the lacrimal glands of mice to establish DES models. The tear secretion rate was estimated by phenol red thread test. Lissamine green eye staining was used to evaluate conjunctival damage. The corneal surface was observed by hematoxylin-eosin (HE) staining and scanning electron microscopy (SEM). The morphology and quantity of conjunctival epithelial cells and goblet cells were observed by Periodic acid-Schiff staining. The expression of NOX4, NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), interleukin-1β (IL-1β), and mucin 5 subtype AC (MUC5AC) was detected by immunohistochemistry. Results: Compared with the Control group, the Model group showed a significant decrease in tear secretion and an upregulation in microscopic image score. The HE staining and SEM showed corneal and conjunctiva damage in the Model group. The protein expression of NOX4, NLRP3, and IL-1β was upregulated, but MUC5AC was downregulated in the Model group. After interfering with NOX4, all these indicators were reversed. Conclusion: The pathological process of concanavalin A-induced DES appears to be related to NOX4.

Background: Diabetes mellitus is associated with increased risk of ocular surface diseases in elderly. We consider neutrophil CD15 as a potential marker of ocular surface damage in type II diabetes mellitus patients., Aim: We aimed to evaluate expression of neutrophil CD15 and correlate it with results of conjunctival impression cytology and routine objective anterior ocular surface tests (TMH, NIBUT, LLT, MGD) in T2DM patients., Materials and Methods: We prospectively enrolled sixty type II diabetes mellitus patients (120 eyes) into a study group. The control group included forty (80 eyes) age- and sex-matched healthy individuals. All patients underwent comprehensive ophthalmological examination, and tear meniscus height test (TMH), noninvasive tear break-up time (NIBUT), lipid layer thickness measurement (LLT), Meibomian gland dysfunction evaluation (MGD), conjunctival impression cytology (CIC) and expression of CD15., Results: Abnormal Nelson's grades of squamous metaplasia (grades 2 and 3) were observed in 50% (60 eyes) of the study group, and 13.8 (11 eyes) of the control group. Fifteen patients with type II diabetes mellitus suffered from grade 3 squamous metaplasia. Nelson's grades of squamous metaplasia have shown a positive correlation with the level of CD15 expression either in the study and control groups (rs = 0.628, p  = <0.0001; rs = 0.746, p  < 0.0001; respectively)., Conclusions: The research shows significantly reduced values of routine objective ocular tests in type II diabetes mellitus patients in comparison to healthy participants older than 60 y.o. Increased CD15 in the peripheral blood is associated with the development of squamous metaplasia and may be used to evaluate the severity of ocular surface damage in type II diabetes mellitus patients., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To establish and characterize a dry eye model in New Zealand rabbits by subcutaneous injections of scopolamine hydrobromide (SCOP)., Methods: Twenty New Zealand male rabbits were injected subcutaneously SCOP for 14 consecutive days; subcutaneous saline was used as a negative control. The correlated clinical parameters of ocular surface dryness were detected in vivo using tear secretion and corneal fluorescein staining. The expression of IL-1β and TNF-α on the ocular surface and in lacrimal glands were analyzed by real-time PCR and western blot on the 14th day. The expression of Mucin-5 subtype AC (MUC5AC) was detected by Immunofluorescence staining in conjunctival tissue., Results: The SCOP-treated rabbits exhibited significantly decreased aqueous tear secretion and increased corneal fluorescein staining scores over time. Both the mRNA expression levels and the protein expression levels of IL-1β and TNF-α were significantly increased after SCOP treatment compared with those after saline treatment. The loss of conjunctival MUC5AC was found in the SCOP-injected rabbits. Some infiltrated inflammatory cells and atrophic acinar cells were observed in the lacrimal gland after SCOP treatment. The disordered structures of the ocular surface and lacrimal glands were also observed., Conclusions: This study showed that repeated subcutaneous SCOP injections successfully elicited some of the typical dry eye symptoms commonly seen in humans.

Conjunctival fibrosis is a common postoperative complication of glaucoma filtration surgery, resulting in uncontrolled intraocular pressure and surgery failure. Therefore, there is an urgent need to understand the molecular mechanisms underlying conjunctival fibrosis and to explore novel pharmacologic anti-fibrosis therapies for glaucoma filtration surgery. Herein, the 4-dimensional data-independent acquisition (4D-DIA) quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/β-catenin pathway in transforming growth factor (TGF)-β1-induced human conjunctival fibroblasts (HConFs). Treatment with ICG-001, a Wnt/β-catenin inhibitor, effectively inhibited cell proliferation and migration in TGFβ1-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix proteins induced by TGFβ1. In addition, ICG-001 reduced the expression level of α smooth muscle actin (α-SMA) and inhibited cell contractility in TGFβ1-treated HConFs. Proteomics data further suggested that αB-crystallin (CRYAB) was a downstream target of Wnt/β-catenin, which was up-regulated by TGFβ1 and down-regulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and β-catenin in TGFβ1-treated HConFs, implying that CRYAB stabilized β-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased extracellular matrix protein levels, and up-regulated α-SMA expression of HConFs under TGFβ1 stimulation. CRYAB rescued TGFβ1-induced fibrotic responses that were suppressed by ICG-001. In conclusion, this study elucidates the regulatory mechanism of the Wnt/β-catenin/CRYAB pathway in conjunctival fibrosis, offering promising therapeutic targets for mitigating bleb scarring after glaucoma filtration surgery., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Linear polyethylenimine (L-PEI) has numerous applications, such as in pharmaceutical formulations, gene delivery, and water treatment. However, due to the presence of secondary amine groups, L-PEI shows a relatively high toxicity and low biocompatibility. Here, various organic anhydrides were used to modify L-PEI to reduce its toxicity and enhance its functionality. We selected methacrylic anhydride, crotonic anhydride, maleic anhydride, and succinic anhydride to modify L-PEI. The structure of the resulting derivatives was characterized using 1 H NMR and FTIR spectroscopies, and their behavior in aqueous solutions was studied using turbidimetric and electrophoretic mobility measurements over a broad range of pHs. A fluorescence flow through method determined the mucoadhesive properties of the polymers to the bovine palpebral conjunctiva. Methacrylated L-PEI and crotonylated L-PEI showed strong mucoadhesive properties at pH 7.4, likely due to covalent bonding with mucin thiol groups. In contrast, maleylated and succinylated L-PEI were poorly mucoadhesive as the pH was above their isoelectric point, resulting in electrostatic repulsion between the polymers and mucin. The toxicity of these polymers was evaluated using in vivo assays with planaria and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H -tetrazolium bromide (MTT) cell viability assay in human alveolar epithelial cells. Moreover, the irritancy of polymers was assessed using a slug mucosa irritation assay. The results demonstrated that anhydride modification mitigated the adverse toxicity effects seen for parent L-PEI.

Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.

The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca 2+ -signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca 2+ -concentration ([Ca 2+ ] i ) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca 2+ ] i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca 2+ ] i induced by C5a. Moreover, the MR-induced rise in Ca 2+ -influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.

Subconjunctival fibrosis is critical to the outcomes of several ophthalmic conditions or procedures, such as glaucoma filtering surgery. This study aimed to investigate the anti-fibrotic effect of celastrol on subconjunctival fibrosis and to further reveal the underlying mechanisms. We used celastrol-loaded nanomicelles hydrogel hybrid as a sustained-release drug. A rabbit model of subconjunctival fibrosis following silicone implantation was used for in vivo study, and TGF-β1-induced human pterygium fibroblast (HPF) activation as an in vitro model. The effects of celastrol on inhibiting TGF-β1-induced migration and proliferation of HPFs were evaluated by scratch wound assay and CCK-8, respectively. Immunofluorescence and western blotting were used to examine the effect of celastrol on the expression of α-SMA, collagen I, fibronectin, and the targets of the Hippo signaling pathway. We found that in vivo celastrol treatment reduced the expression of YAP and TAZ in subconjunctival tissue. Moreover, celastrol alleviated collagen deposition and subconjunctival fibrosis at 8 weeks. No obvious tissue toxicity was observed in the rabbit models. Mechanistically, celastrol significantly inhibited TGF-β1-induced proliferation and migration of HPFs. Pretreatment of HPFs with celastrol also suppressed the TGF-β1-induced protein expression of α-SMA, collagen I, fibronectin, TGF-βRII, phosphorylated Smad2/3, YAP, TAZ, and TEAD1. In conclusion, celastrol effectively prevented subconjunctival fibrosis through inhibiting TGF-β1/Smad2/3-YAP/TAZ pathway. Celastrol could serve as a promising therapy for subconjunctival fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: The purpose of this study was to determine the association between corneal images provided by in vivo confocal microscopy (IVCM) with clinical parameters and conjunctival expression of HLA-DR antigen in patients with dry eye disease (DED)., Methods: Two hundred fourteen eyes of 214 patients with DED were analyzed, consisting of 2 groups of patients - 63 with autoimmune dry eye disease (AIDED) and 151 with non-autoimmune dry eye disease (NAIDED). Patients underwent a full clinical examination, including symptom screening, using the Ocular Surface Disease Index (OSDI) questionnaire, and objective analysis of DED signs by Schirmer's testing, tear break-up time (TBUT), Oxford's test, and IVCM corneal imaging. The IVCM scoring criteria were based on corneal sub-basal nerve density (ND), nerve morphology (NM), and inflammatory cell (IC) density. Quantification of conjunctival HLA-DR antigen was performed by flow cytometry., Results: The total IVCM score (T-IVCM) as well as the IVCM-IC subscore (sc) were positively correlated with HLA-DR levels with r = 0.3, P < 0.001 and r = 0.3, P < 0.01, respectively in the total population of patients with DED. The IVCM-NDsc was negatively correlated with TBUT in patients with AIDED (r = -0.2, P < 0.05) and with the Schirmer's test in patients with NAIDED (r = -0.24, P < 0.05). However, the IVCM-NMsc was positively correlated with the Oxford score only in patients with AIDED (r = 0.3, P < 0.05)., Conclusions: The proposed IVCM scoring system showed significant correlations with clinical parameters along with conjunctival HLA-DR quantification in patients with DED., Translational Relevance: The IVCM grading score represents an interesting point of commonality among clinical parameters, imaging, and molecular investigation of the ocular surface.

The ocular system is in constant interaction with the environment and with numerous pathogens. The ATP-binding cassette (ABC) transporters represent one of the largest groups among the transmembrane proteins. Their relevance has been demonstrated for their defense function against biotic and abiotic stress factors, for metabolic processes in tumors and for their importance in the development of resistance to drugs. The aim of this study was to analyze which ABC transporters are expressed at the ocular surface and in the human lacrimal apparatus. Using RT-PCR, all ABC transporters known to date in humans were examined in tissue samples from human cornea, conjunctiva, meibomian glands and lacrimal glands. The RT-PCR analyses revealed the presence of all ABC transporters in the samples examined, although the results for some of the 48 transporters known in human and analyzed were different in the various tissues. The present results provide information on the expression of ABC transporters at the mRNA level on the ocular surface and in the lacrimal system. Their detection forms the basis for follow-up studies at the protein level, which will provide more information about their physiological significance at the ocular surface and in the lacrimal system and which may explain pathological effects such as drug resistance., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Benedikt Kleinsasser reports financial support was provided by Sicca Forschungsförderung of the professional Association of German Ophthalmologists. Martin Schicht reports financial support was provided by Sicca Forschungsförderung of the professional Association of German Ophthalmologists. Martin Schicht reports financial support was provided by Ernst und Berta Grimmke Stiftung. Martin Schicht reports financial support was provided by Ernst Muck Stiftung. Friedrich Paulsen reports financial support was provided by German Research Foundation. Friedrich Paulsen is Editor-in-Chief of the Annals of Anatomy If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Pterygium is a frequent eye surface condition that is characterized by a high rate of proliferation, fibrovascular development, cellular migration, corneal infiltration, and angiogenesis. We investigated that ex vivo primary pterygium and conjunctival cell cultures were generated to analyze the effect of trehalose on cellular proliferation. After trehalose treatment, we performed microarray analysis to evaluate changes in the mRNA profile. We analyzed gene ontology (GO) and KEGG pathways to identify hub genes that changed expression levels after treatment and were associated with pterygium development. We selected three genes to verify their expression levels using qRT-PCR. The study also evaluated the impact of trehalose treatment on cell migration through a wound-healing assay. Our results suggested that pterygium cell proliferation was inhibited in a dose-dependent manner by trehalose. 2354 DEG were identified in pterygium and conjunctiva cells treated with trehalose compared to untreated groups. Functional enrichment analysis showed that differentially expressed mRNAs are involved in proliferation, vasculature development, and cell migration. We identified ten hub genes including upregulated (RANBP3L, SLC5A3, RERG, ANKRD1, DHCR7, RAB27B, GPRC5B, MSMO1, ASPN, DRAM1) and downregulated (TNC, PTGS2, GREM2, NPTX1, NR4A1, HMOX1, CXCL12, IL6, MYH2, TXNIP). Microarray analysis and functional investigations suggest that trehalose affects the pathogenesis of pterygium by modifying the expression of genes involved in crucial pathways related to cell function., (© 2024 The Author(s). Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

Objectives: To examine changes in tear oxidative stress levels and tear film functions in patients with blepharoptosis and dermatochalasis following conjunctiva-Müller muscle resection (CMMR) and blepharoplasty surgeries., Materials and Methods: This prospective study included 32 healthy controls and 62 patients with blepharoptosis or dermatochalasis. CMMR surgery was performed in 20 eyes and upper blepharoplasty was performed in 42 eyes. Tear oxidative stress markers (8-hydroxy-2’-deoxyguanosine [8-OHdG] and 4-hydroxy-2-nonenal [4-HNE]) were quantified by enzyme-linked immunosorbent assay and tear film functions were evaluated preoperatively and at 1 and 6 months postoperatively. The same assessments were performed in the control group at the same time points., Results: Preoperative tear 8-OHdG and 4-HNE levels were lower in healthy controls (52.8±13.5 ng/mL and 27.8±6.4 ng/mL, respectively) compared to patients with dermatochalasis (86.1±37.2 ng/mL and 29.8±11.1 ng/mL, respectively) and blepharoptosis (90.4±39.3 ng/mL and 43.1±4.2 ng/mL, respectively) (p<0.001). 8-OHdG levels were increased at 1 month after CMMR, while both markers were decreased 1 month postoperatively in the blepharoplasty group (p=0.034). Schirmer 1 and OSDI scores did not change throughout the visits in both patient groups, but a temporary decrease in tear break-up time (TBUT) was observed after CMMR (p=0.017)., Conclusion: Dermatochalasis and blepharoptosis were associated with higher tear oxidative stress levels. CMMR surgery caused a temporary decrease in TBUT scores and an increase in oxidative stress in the first postoperative month., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors., (©Copyright 2024 by the Turkish Ophthalmological Association / Turkish Journal of Ophthalmology published by Galenos Publishing House.)

Tear matrix metalloproteinase (MMP)-9 is an inflammatory signal in patients with dry eye (DE). In the present study, to understand the action mechanism of probiotic LB101 (Lactobacillus plantarum NK151 and Bifidobacterium bifidum NK175 [4:1] mix) against DE, we investigated its effect on tear amount and inflammatory marker expression levels in mice with unilateral exorbital lacrimal gland excision/atropine-benzalkonium chloride application (EB) or fecal microbiota transplantation from mice with EB (eFMT). Oral gavage of LB101 increased EB-suppressed tear amount and decreased EB-induced blinking number. Furthermore, LB101 decreased EB-induced TNF-α, IL-1β, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva, while EB-suppressed IL-10 and occludin expression increased. LB101 also decreased EB-induced TNF-α and IL-1β expression and NF-κB+CD11c+ cell population in the colon. eFMT also decreased tear amount and increased blinking number in the transplanted mice. eFMT increased TNF-α, IL-1β, and MMP-9 expression and TNF-α+ and NF-κB+CD11c+ cell populations in the conjunctiva and TNF-α and IL-1β expression and NF-κB+CD11c+ cell populations in the colon. Oral gavage of LB101 increased eFMT-suppressed tear amount and decreased eFMT-induced blinking number. Furthermore, LB101 decreased TNF-α, IL-1β, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva and TNF-α and IL-1β expression and NF-κB+CD11c+ cell population in the colon, while eFMT-suppressed IL-10 and occludin expression decreased. Furthermore, LB101 increased eFMT-suppressed Muribaculaceae, Prevotellaceae, and Lactobacillaceae populations in the gut microbiota, while eFMT-induced Bacteroidaceae population decreased. These findings suggest that DE may cause gut dysbiosis, which may be a risk factor for DE, and LB101 may alleviate DE with gut inflammation by suppressing the expression of MMP-9 and proinflammatory cytokines TNF-α and IL-1β with the regulation of gut microbiota-involved NF-κB signaling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although glaucoma is a leading cause of irreversible blindness worldwide, its pathogenesis is incompletely understood, and intraocular pressure (IOP) is the only modifiable risk factor to target the disease. Several associations between the gut microbiome and glaucoma, including the IOP, have been suggested. There is growing evidence that interactions between microbes on the ocular surface, termed the ocular surface microbiome (OSM), and tear proteins, collectively called the tear proteome, may also play a role in ocular diseases such as glaucoma. This study aimed to find characteristic features of the OSM and tear proteins in patients with glaucoma. The whole-metagenome shotgun sequencing of 32 conjunctival swabs identified Actinobacteria, Firmicutes, and Proteobacteria as the dominant phyla in the cohort. The species Corynebacterium mastitidis was only found in healthy controls, and their conjunctival microbiomes may be enriched in genes of the phospholipase pathway compared to glaucoma patients. Despite these minor differences in the OSM, patients showed an enrichment of many tear proteins associated with the immune system compared to controls. In contrast to the OSM, this emphasizes the role of the proteome, with a potential involvement of immunological processes in glaucoma. These findings may contribute to the design of new therapeutic approaches targeting glaucoma and other associated diseases.

Purpose: This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease., Methods: A dry eye model was established in C57BL/6 mice exposed to desiccating stress (DS) with untreated (UT) mice as controls. DS mice were topically treated with 2.0% ectoine or PBS vehicle. Corneal epithelial defects were assessed by Oregon Green Dextran (OGD) fluorescent staining. Conjunctival goblet cells, ocular mucins, and T help (Th) cytokines were evaluated by immunofluorescent staining or ELISA, and RT-qPCR., Results: Compared with UT mice, corneal epithelial defects were detected as strong punctate OGD fluorescent staining in DS mice with vehicle, whereas ectoine treatment largely reduced OGD staining to near-normal levels. Conjunctival goblet cell density and cell size decreased markedly in DS mice, but was significantly recovered by ectoine treatment. The protein production and mRNA expression of two gel-forming secreted MUC5AC and MUC2, and 4 transmembrane mucins, MUC1, MUC4, MUC16, and MUC15, largely decreased in DS mice, but was restored by ectoine. Furthermore, Th2 cytokine IL-13 was inhibited, whereas Th1 cytokine IFN-γ was stimulated at protein and mRNA levels in conjunctiva and draining cervical lymph nodes (CLNs) of DS mice, leading to decreased IL-13/IFN-γ ratio. Interestingly, 2.0% ectoine reversed their alternations and restored IL-13/IFN-γ balance., Conclusions: Our findings demonstrate that topical ectoine significantly reduces corneal damage, and enhances goblet cell density and mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This suggests therapeutic potential of natural osmoprotectant ectoine for dry eye disease.

Purpose: Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring. In advanced stage AAK, typically, conjunctival epithelial cells slowly replace the corneal epithelium. Based on previous results we hypothesize that alterations of the conjunctival cells in congenital aniridia may also support the corneal conjunctivalization process. The aim of this study was to identify deregulated proteins in conjunctival impression cytology samples of congenital aniridia subjects., Methods: Conjunctival impression cytology samples of eight patients with congenital aniridia [age 34.5 ± 9.9 (17-51) years, 50% female] and eight healthy subjects [age 34.1 ± 11.9 (15-54) years, 50% female] were collected and analysed using mass spectrometry. Proteomic profiles were analysed in terms of molecular functions, biological processes, cellular components and pathway enrichment using the protein annotation of the evolutionary relationship (PANTHER) classification system., Results: In total, 3323 proteins could be verified and there were 127 deregulated proteins (p < 0.01) in congenital aniridia. From the 127 deregulated proteins (DEPs), 82 altered biological processes, 63 deregulated cellular components, 27 significantly altered molecular functions and 31 enriched signalling pathways were identified. Pathological alteration of the biological processes and molecular functions of retinol binding and retinoic acid biosynthesis, as well as lipid metabolism and apoptosis related pathways could be demonstrated., Conclusions: Protein profile of conjunctival impression cytology samples of aniridia subjects identifies alterations of retinol binding, retinoic acid biosynthesis, lipid metabolism and apoptosis related pathways. Whether these changes are directly related to PAX6 haploinsufficiency, must be investigated in further studies. These new findings offer the possibility to identify potential new drug targets., (© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: The study aims to characterize the robustness of distinct clinical assessments in identifying the underlying conditions of dry eye disease (DED), with a specific emphasis on the involvement of conjunctival goblet cells., Methods: Seven rabbits receiving surgical removal of the lacrimal and Harderian glands were divided into two groups, one with ablation of conjunctival goblet cells by topical soaking of trichloroacetic acid (TCA) to the bulbar conjunctiva ( n  = 3) and one without ( n  = 4), and the conditions of DED were assessed weekly using Schirmer test, tear breakup time (TBUT), tear osmolarity, and National Eye Institute (NEI) fluorescein staining grading. After 8 weeks, the rabbits were sacrificed, and the eyes were enucleated for histopathological examination., Results: Histopathological analysis revealed corneal epithelial thinning in both groups. While TCA soaking significantly decreased the density of conjunctival goblet cells, DED rabbits without TCA also showed a partial reduction in goblet cell density, potentially attributable to dacryoadenectomy. Both groups showed significant decreases in Schirmer test and TBUT, as well as an increase in tear osmolarity. In DED rabbits with TCA soaking, tear osmolarity increased markedly, suggesting that tear osmolarity is highly sensitive to loss and/or dysfunction of conjunctival goblet cells. Fluorescein staining was gradually and similarly increased in both groups, suggesting that fluorescein staining may not reveal an early disruption of the tear film until the prolonged progression of DED., Conclusion: The Schirmer test, TBUT, tear osmolarity, and NEI fluorescein grading are distinct, yet complementary, clinical assessments for the evaluation of DED. By performing these assessments in definitive DED rabbit models, both with and without ablation of conjunctival goblet cells, the role of these cells in the homeostasis of tear osmolarity is highlighted. Characterizing the robustness of these assessments in identifying the underlying conditions of DED will guide a more appropriate management for patients with DED.

The prevalence of Light-emitting diodes (LEDs) has exposed us to an excessive amount of blue light (BL) which causes various ophthalmic diseases. Previous studies have shown that conjunctiva is vulnerable to BL. In this study, we aimed to investigate the underlying mechanism of BL-induced injury in conjunctiva. We placed C57BL/6 mice and human conjunctival epithelial cell lines (HCECs) under BL (440 nm ± 15 nm, 0.2 mW/cm 2 ) to establish a BL injury model in vivo and in vitro. Immunohistochemistry and MDA assay were used to identify lipid peroxidation (LPO) in vivo. HE staining was applied to detect morphological damage of conjunctival epithelium. DCFH-DA, C11-BODIPY 581/591, Calcein-AM, and FeRhoNox™-1 probes were performed to identify ferroptosis levels in vitro. Real-time qPCR and Western blotting techniques were employed to uncover signaling pathways of blue light-induced ferroptosis. Our findings demonstrated that BL affected tear film instability and induced conjunctival epithelium injury in vivo. Ferrostatin-1 significantly alleviated blue light-induced ferroptosis in vivo and in vitro. BL downregulates the levels of solute carrier family 7 member 11 (SLC7A11), Ferritin heavy chain (FTH1), and glutathione peroxidase (GPX4) by inhibiting the activation and translocation of the Signal transducer and activator of transcription 3 (STAT3) from inducing Fe 2+ burst, ROS and LPO accumulation, ultimately resulting in ferroptosis. This study will offer new insight into BL-induced conjunctival injury and LED-induced dry eye., Competing Interests: Declaration of competing interest There are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The tissues of the integument covering the ocular surface comprise a mucus membrane functioning as a protective physical barrier and has the ability to mount a defensive inflammatory response. Since lipid metabolism has a role in both of these functions, we studied normal membrane phospholipids (PL) of the cornea and bulbar conjunctiva to (1) determine baseline PL profiles of these tissues, (2) compare and contrast these individual PL metabolite profiles as well as groups of metabolites, and (3) describe pathway-specific metabolic interrelations among these tissues. Corneal and conjunctival tissue samples were isolated from rabbit eyes (n = 30) and extracted with chloroform-methanol using a modified Folch procedure. 31 P nuclear magnetic resonance spectroscopy was used to qualitatively and quantitatively measure tissue PL profiles. The cornea and conjunctiva, respectively, have the following PL composition (mole % of total detected phospholipid): phosphatidylglycerol (PG) -, 0.4; lysophosphatidylethanolamine 1.2, -; phosphatidic acid -, 0.4; diPG (cardiolipin) 2.1, 3.5; unknown PL at the chemical shift of 0.13 δ 1.5, 0.9; ethanolamine plasmalogen 11.2, 13.0; phosphatidylethanolamine 11.5, 12.8; phosphatidylserine 8.9, 10.1; sphingomyelin 10.2, 10.7; lysophosphatidylcholine 0.9, 1.4; phosphatidylinositol 5.3, 5.3; phosphatidylcholine (PC) plasmalogen or alkylacylPC 2.2, 1.9; PC 45.1, 40.0. In addition, 28 PL metabolic indices were calculated from these data, which permitted pathway-specific lipid analyses. This study (1) establishes PL profiles of the two ocular tissues of the integument that cover the surface of the eye, (2) compares and contrasts indices comprised of ratios and combinations of PL, and (3) describes pathway-specific metabolic interrelations among these tissues to serve as baselines for studies involving the distribution of tissue phospholipids., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bioinformatics analysis was performed to reveal the underlying pathogenesis of type 2 diabetes (T2DM) dry eye(DE) and to predict the core targets and potential pathways for electroacupuncture (EA) treatment of T2DM DE, in which key targets such as Toll-likereceptor4 (TLR4), NF-κB and Tumor necrosis factor-α (TNF-α) may be involved. Next, streptozotocin and a high-fat diet were used to generate T2DM-DE rats. Randomly picked EA, fluorometholone, model, and sham EA groups were created from successfully modelled T2DM DE rats. Six more rats were chosen as the blank group from among the normal rats. The results of DE index showed that EA improved the ocular surface symptoms.HE staining showed that EA attenuated the pathological changes in the cornea, conjunctiva and lacrimal gland of T2DM DE rats. EA decreased the expression of TLR4, MyD88, P-NF-κB P65, and TNF-α in the cornea, conjunctiva, and lacrimal gland, in accordance with immunofluorescence and Western blot data. Thus, EA reduced ocular surface symptoms and improved pathological changes of cornea, conjunctiva, and lacrimal gland induced by T2DM DE inT2DM DE rats, and the mechanism may be related to the inhibition of overactivation of the TLR4/NF-κB signaling pathway by EA and thus attenuating ocular surface inflammation.

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The purpose of this study is to evaluate loose suture-related inflammation and activation of conjunctiva-associated lymphoid tissue (CALT) in patients after keratoplasty. The patients who were treated with keratoplasty at the First Affiliated Hospital of Harbin Medical University between 2015 and 2022 were recruited into the study. We evaluated the time and location of loose suture development in patients after keratoplasty. In addition, in vivo confocal microscopy was used to evaluate the activation of CALT and the accumulation of inflammatory cells around loose sutures. Meso Scale Discovery assay detection kits were used to evaluate the inflammatory cytokines in the tears of patients before and after the loose suture was removed. In this study, we collected the information from 212 cases (212 eyes) who had PK (126 eyes) and DALK-treated (86 eyes) for corneal transplantation, including 124 males and 88 females, aged 14-84 years old. The average age was 50.65 ± 16.81 years old. Corneal sutures were more prone to loose at 3 months and 6 months after keratoplasty, and the frequent sites were at 5 and 6 o'clock. An increased number of inflammatory cells could be observed around the loose sutures than normal sutures (P < 0.001). In CALT, the density of diffuse lymphocytes (P < 0.001), follicles (P < 0.001), and parafollicular lymphocytes (P < 0.001) were higher and the central reflection of the follicles (P < 0.001) was stronger when suture loosening happened. The levels of inflammatory cytokines such as IL-1β (P = 0.003), IL-8 (P = 0.012), and TNF-α (P < 0.001) were higher in the tears of the patients with loose sutures. The activation of CALT was partly settled after removing the loose sutures. In conclusion, loose sutures after corneal transplantation can lead to increased infiltration of inflammatory cells, activation of CALT, and increased secretion of inflammatory cytokines in the tears of patients. Regular follow-up to identify and solve the problem in time can avoid suture-related complications., (© 2024. The Author(s).)

Purpose: Pterygium is a common ocular surface disease defined by fibrovascular conjunctival growth extending onto the cornea. However, its pathogenesis remains unclear. This study aimed to determine the role of CD44, proliferating cell nuclear antigen (PCNA), and E-cadherin in pterygium formation and recurrence., Methods: Sixty patients with pterygium participated in the study, and we collected conjunctival samples from 30 patients to form a control group. CD44, PCNA, and E-cadherin expressions in surgically excised pterygium were compared with tissue samples from the control group., Results: We observed that the percentages of CD44 and PCNA were statistically higher in the primary pterygium group and recurrent pterygium group than in the control group (P < 0.001 and P < 0.001, respectively). Conversely, E-cadherin values were statistically higher in the control group than in the primary and recurrent pterygium groups (P = 0.013 and P < 0.001, respectively)., Conclusion: Cell proliferation and cell adhesion factors may play important roles in the pathogenesis of pterygium., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

A pterygium is a common conjunctival degeneration and inflammatory condition. It grows onto the corneal surface or limbus, causing blurred vision and cosmetic issues. Ultraviolet is a well-known risk factor for the development of a pterygium, although its pathogenesis remains unclear, with only limited understanding of its hereditary basis. In this study, we collected RNA-seq from both pterygial tissues and conjunctival tissues (as controls) from six patients (a total of twelve biological samples) and retrieved publicly available data, including eight pterygium samples and eight controls. We investigated the intrinsic gene regulatory mechanisms closely linked to the inflammatory reactions of pterygiums and compared Asian (Korea) and the European (Germany) pterygiums using multiple analysis approaches from different perspectives. The increased expression of antioxidant genes in response to oxidative stress and DNA damage implies an association between these factors and pterygium development. Also, our comparative analysis revealed both similarities and differences between Asian and European pterygiums. The decrease in gene expressions involved in the three primary inflammatory signaling pathways-JAK/STAT, MAPK, and NF-kappa B signaling-suggests a connection between pathway dysfunction and pterygium development. We also observed relatively higher activity of autophagy and antioxidants in the Asian group, while the European group exhibited more pronounced stress responses against oxidative stress. These differences could potentially be necessitated by energy-associated pathways, specifically oxidative phosphorylation.

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5-10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface., (© 2024. The Author(s).)

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears., Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1β, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay., Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance., Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alenezi, Parnell, Schibeci, Ozkan, Willcox, White and Carnt.)

Purpose: Pterygium is a vision-threatening conjunctival fibrovascular degenerated disease with a high global prevalence up to 12 %, while no absolute pharmacotherapy has been applied in clinics. In virtue of single-cell RNA sequencing (scRNA-seq) technique, our study investigated underlying pathogeneses and potential therapeutic targets of pterygium from the cellular transcriptional level., Methods: A total of 45605 cells from pterygium of patients and conjunctiva of normal controls (NC) were conducted with scRNA-seq, and then analyzed via integrated analysis, pathway enrichment, pseudotime trajectory, and cell-cell communications. Besides, immunofluorescence and western blot were performed in vivo and in vitro to verify our findings., Results: In brief, 9 major cellular types were defined, according to canonical markers. Subsequently, we further determined the subgroups of each major cell lineages. Several newly identified cell sub-clusters could promote pterygium, including immuno-fibroblasts, epithelial mesenchymal transition (EMT)-epithelial cells, and activated vascular endothelial cells (activated-vEndo). Besides, we also probed the enrichment of immune cells in pterygium. Particularly, macrophages, recruited by ACKR1 + activated-vEndo, might play an important role in the development of pterygium by promoting angiogenesis, immune suppression, and inflammation., Conclusion: An intricate cellular niche was revealed in pterygium via scRNA-seq analysis and the interactions between macrophages and ACKR1 + activated-vEndo might be the key part in the development of pterygia., Competing Interests: Declaration of competing interest We declared that there was no conflict of interest in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Allergic conjunctivitis (AC), an allergen-induced ocular inflammatory disease, primarily involves mast cells (MCs) and eosinophils. The role of neuroimmune mechanisms in AC, however, remains to be elucidated. We investigated the effects of transient receptor potential vanilloid 1 (TRPV1)-positive sensory nerve ablation (using resiniferatoxin) and TRPV1 blockade (using Acetamide, N-[4-[[6-[4-(trifluoromethyl)phenyl]-4-pyrimidinyl]oxy]-2-benzothiazolyl] (AMG-517)) on ovalbumin-induced conjunctival allergic inflammation in mice. The results showed an exacerbation of allergic inflammation as evidenced by increased inflammatory gene expression, MC degranulation, tumor necrosis factor-α production by MCs, eosinophil infiltration and activation, and C-C motif chemokine 11 (CCL11) (eotaxin-1) expression in fibroblasts. Subsequent findings demonstrated that TRPV1 + sensory nerves secrete somatostatin (SST), which binds to SST receptor 5 (SSTR5) on MCs and conjunctival fibroblasts. SST effectively inhibited tumor necrosis factor-α production in MCs and CCL11 expression in fibroblasts, thereby reducing eosinophil infiltration and alleviating AC symptoms, including eyelid swelling, lacrimation, conjunctival chemosis, and redness. These findings suggest that targeting TRPV1 + sensory nerve-mediated SST-SSTR5 signaling could be a promising therapeutic strategy for AC, offering insights into neuroimmune mechanisms and potential targeted treatments., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)