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Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Bacterial keratitis is a common cause of blindness. Antibiotic treatment leads to the rapid release of lipopolysaccharide (LPS), which can activate corneal fibroblasts and cause persistent and excessive inflammatory responses. The anti-inflammatory drugs currently used to treat keratitis have serious side effects. Therefore, the ability of sodium butyrate (NaB), which can suppress the production of proinflammatory cytokines and promote the production of anti-inflammatory cytokines, to ameliorate keratitis was assessed in the present study., Methods: The effect of NaB on the viability of primary human corneal fibroblasts was assayed with a CCK-8 kit. Cell migration was assessed by an in vitro scratch assay. Cell phenotypes were assessed by Western blotting and immunofluorescence staining. An antibody array was used to measure the production of proinflammatory cytokines and chemokines., Results: At 0-1 mM, NaB had no significant effect on cell viability, promoted the expression of the keratocyte marker keratocan and inhibited the fibroblast marker vimentin. Inhibition of cell migration was observed in the wound healing assay. By targeting the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway, NaB decreased the levels of inflammation-related cytokines and chemokines whose expression was induced by LPS., Conclusions: NaB maintained the keratocyte phenotype, inhibited cell migration, and relieved LPS-induced inflammatory responses through the JAK/STAT signalling pathway., 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.)

Background: Human artificial corneas (HAC) generated by tissue engineering recently demonstrated clinical usefulness in the management of complex corneal diseases. However, the biological mechanisms associated to their regenerative potential need to be elucidated., Methods: In the present work, we generated HAC using nanostructured fibrin-agarose biomaterials with cultured corneal epithelial and stromal cells, and we compared the structure and histochemical and immunohistochemical profiles of HAC with control native corneas (CTR-C) and limbus (CTR-L) to determine the level of biomimicry of the HAC with these two native organs., Results: HAC tissues consisted of a stratified epithelium and a cellular stromal substitute. The interface between stroma and epithelium was similar to that of CTR-C, without the finger-shaped palisades of Vogt found in CTR-L, and contained a poorly developed basement membrane as determined by PAS histochemistry. Analysis of the stromal layer revealed that HAC contained significantly lower amounts of extracellular matrix components (collagen, proteoglycans, decorin, keratocan, and lumican) than CTR-C and CTR-L, with all samples being devoid of elastic and reticular fibers. At the epithelial level, HAC were strongly positive for several cytokeratins, although KRT5 was lower in HAC as compared to CTR-C and CTR-L. The expression of crystallin lambda was lower in HAC than in control tissues, whereas crystallin alpha-a was similar in HAC and CTR-C. No differences were found among HAC and controls for the cell-cell junction proteins CX43 and TJP1. When specific markers were analyzed, we found that HAC expression profile of KRT3, KRT19, KRT15, and ΔNp63 was more similar to CTR-L than to CTR-C., Conclusions: These results suggest that HAC generated in the laboratory could be structurally and functionally more biomimetic to the structure found at the corneal limbus than to the central cornea, and open the door to the use of these artificial tissues in patients with limbal deficiency., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm -1 . The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 cornea serves as a vital protective shield for the eye, safeguarding its intricate internal structures from external threats. Damage to the cornea compromises this protective function, triggering inflammation and potentially causing long-term harm. While ginsenoside Rk3 has demonstrated potential for repairing the corneal barrier and reducing inflammation, its effectiveness in treating corneal damage remains relatively unexplored. This comprehensive study uses both in vivo and in vitro models to investigate the therapeutic capabilities of ginsenoside Rk3. Using two models of corneal damage, a benzalkonium chloride-induced mouse model and a high osmolarity-induced human corneal epithelial cell model, we scrutinized the effects of ginsenoside Rk3 treatment. Our results showed that ginsenoside Rk3-treated mice manifested reduced corneal damage and inflammation compared with their untreated counterparts. Furthermore, mice treated with ginsenoside Rk3 exhibited an organized arrangement of corneal cells and diminished stromal layer thickness, indicating reparative properties of ginsenoside Rk3. Additionally, ginsenoside Rk3 increased the expression of tight junction proteins, suppressed inflammatory factors, and decreased HMGB1 protein expression, thereby modulating downstream signaling pathways. Collectively, our findings present compelling evidence that ginsenoside Rk3 is a promising therapeutic option for corneal injury. By repairing the corneal barrier, mitigating inflammation, and modulating specific protein levels, ginsenoside Rk3 opens new avenues for managing corneal damage.

Corneal blindness affects more than 5 million individuals, with over 180,000 corneal transplantations (CTs) performed annually. In high-risk CTs, almost all grafts are rejected within 10 years. Here, we investigated adeno-associated virus (AAV) ex vivo gene therapy to establish immune tolerance in the corneal allograft to prevent high-risk CT rejection. Our previous work has demonstrated that HLA-G contributes to ocular immune privilege by inhibiting both immune cells and neovascularization; however, homodimerization is a rate-limiting step for optimal HLA-G function. Therefore, a chimeric protein called single-chain immunomodulator (scIM), was engineered to mimic the native activity of the secreted HLA-G dimer complex and eliminate the need for homodimerization. In a murine corneal burn model, AAV8-scIM significantly reduced corneal vascularization and fibrosis. Next, ex vivo AAV8-scIM gene delivery to corneal allografts was evaluated in a high-risk CT rejection rabbit model. All scIM-treated corneas were well tolerated and transparent after 42 days, while 83% of vehicle-treated corneas were rejected. Histologically, AAV-scIM-treated corneas were devoid of immune cell infiltration and vascularization, with minimal fibrosis at the host-graft interface. The data collectively demonstrate that scIM gene therapy prevents corneal neovascularization, reduces trauma-induced corneal fibrosis, and prevents allogeneic CT rejection in a high-risk large animal model., Competing Interests: Declaration of interests M.L.H. and R.B.S. are co-inventors of the scIM technology evaluated here and are listed on a patent (pending) owned by Texas Tech University and UNC-Chapel Hill. M.L.H., R.B.S., C.L., and B.C.G. are co-founders of Bedrock Therapeutics, which has licensed the scIM technology evaluated here., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate the ocular distribution of omidenepag isopropyl (OMDI) and its active form omidenepag (OMD), an EP2 receptor agonist, after topical administration of OMDI into rabbit and monkey eyes, and to determine whether OMDI and OMD interact with target receptors or enzymes of other antiglaucoma agents., Methods: Both eyes of six rabbits and of 14 monkeys were topically instilled with 0.03% [14C]-OMDI. Rabbits were sacrificed after one to four hours, and ocular tissues were collected. Monkeys were sacrificed after 0.25 to 24 hours, and blood and ocular tissues were collected. Radioactivity was measured in each sample. The interactions of OMDI and OMD with the receptors and enzymes associated with the mechanisms of action of other antiglaucoma agents were evaluated., Results: Most radioactivity applied to rabbit eyes was recovered as OMD from the cornea, aqueous humor, and iris-ciliary body. Similarly, high concentrations of radioactivity were observed in monkey cornea, bulbar/palpebral conjunctiva, and trabecular meshwork. OMD bound to EP2 receptors, but neither OMD nor OMDI bound to α2A, β1, and β2 adrenergic receptors or inhibited enzymatic activities of CA1 and CA2. OMD and OMDI had little or no effect on ROCK1 and ROCK2., Conclusions: OMDI rapidly permeates rabbit and monkey corneas and is converted to OMD, which distributes into anterior ocular tissues. Neither OMD nor OMDI interacted with the target receptors or enzymes of other antiglaucoma agents, suggesting that OMD interacts highly selectively with EP2 receptors., Translational Relevance: OMDI is a specific antiglaucoma agent that interacts selectively with ocular EP2 receptors.

Purpose: To determine whether γδ T cells regulate natural killer (NK) cells in the herpes simplex virus 1 (HSV-1)-infected cornea., Methods: CD57Bl/6 (wild-type [WT]), TCRδ-/-, and IFN-γ-/- mice were infected intracorneally with HSV-1. TCR-/- mice were treated with IL-17A at 24 hours post-infection (PI), and the WT mice received treatments of fingolimod (FTY720) and anti-IL-17A. At 48 hours PI, corneas were excised, and intracellular staining flow cytometry was performed, as well as multiplex analysis. Additionally, single-cell RNA sequencing (scRNAseq) was done to analyze the transcriptome of NK cells from WT and TCRδ-/- mice., Results: In mice lacking γδ T cells, there were significantly fewer NK cells following ocular HSV-1 infection. This reduction of NK cells corresponded with lower levels of cytokines and chemokines associated with the antiviral response. Furthermore, NK cells from WT mice had enriched IL-17A signaling compared to those from TCRδ-/- mice. The NK cell response was partially rescued in TCRδ-/- mice by administration of IL-17A. Correspondingly, the NK cell response could be blunted in WT mice by administration of anti-IL-17A. Finally, IFN-γ-/- mice had significantly less IL-17A production compared to WT mice., Conclusions: γδ T17 cells promote NK cell accumulation in HSV-1-infected corneas. In turn, NK cells secrete IFN-γ, which negatively regulates further IL-17A production by γδ T cells.

Different therapeutic modalities, including steroids, have been used to treat corneal scarring. However, the ability of steroids to reduce corneal scarring is limited and associated with numerous side effects. Our previous studies have demonstrated that topical hepatocyte growth factor (HGF) after corneal injury suppresses the development of stromal scars. Here, we investigated whether HGF can re-establish corneal clarity and normalize tissue structure in corneas with pre-existing scars. Corneal scarring was induced by mechanically removing the corneal epithelium and the anterior third of the stroma using a hand-held Algerbrush II in C57BL/6 mice. Substantial scar tissue formed by day 10 post-injury, at which time the epithelium was debrided and treated with 0.1% recombinant mouse HGF, 0.1% dexamethasone (steroid) or 0.1% control protein thrice a day for 10 days. Corneal clarity was significantly restored in the HGF treatment group, compared to both the steroid and control protein treatment groups. Moreover, HGF treatment downregulated the expression of αSMA and upregulated the expression of extracellular matrix-remodeling matrix metalloproteinases 1 and 10 (MMP1 and MMP10), suggesting HGF upregulates tissue remodeling molecule MMP1 and 10 to promote tissue restoration. These findings offer novel insights into the mechanisms by which HGF re-establishes corneal clarity, and promotes epithelial regeneration in corneas with pre-existing stromal scarring., (© 2024. The Author(s).)

Rationale: The cornea is a unique structure that maintains its clarity by remaining avascular. Corneal injuries can lead to neovascularisation (CNV) and fibrosis and are the third most common cause of blindness worldwide., Objective: Corneal injuries induce an immune cell infiltration to initiate reparative processes. However, inflammation caused by sustained immune cell infiltration is known to be detrimental and can delay the healing process. This study was designed to understand the potential role of neutrophil and epithelial cell crosstalk in post-injury CNV., Methods and Results: Western blotting and immunostaining assays demonstrated that neutrophils infiltrated corneas and underwent pyroptosis following acute alkali injury. In vivo studies showed that genetic ablation of Gasdermin D (GsdmD), a key effector of pyroptosis, enhanced corneal re-epithelialisation and suppressed post-injury CNV. In vitro co-culture experiments revealed that interleukin-1β (IL-1β) was released from pyroptotic neutrophils which suppressed migration of murine corneal epithelial cells. Real-time RT-PCR and immunostaining assays identified two factors, Wnt5a and soluble fms-like tyrosine kinase-1 (sflt-1), highly expressed in newly healed epithelial cells. sflt-1 is known to promote corneal avascularity. Bone marrow transplantation, antibody mediated neutrophil depletion, and pharmacological inhibition of pyroptosis promoted corneal wound healing and inhibited CNV in an in vivo murine corneal injury model., Conclusion: Taken together, our study reveals the importance of neutrophil/epithelium crosstalk and neutrophil pyroptosis in response to corneal injuries. Inhibition of neutrophil pyroptosis may serve as a potential treatment to promote corneal healing without CNV., Key Points: Neutrophil pyroptosis delays re-epithelialization after corneal injury Compromised re-epithelialization promotes corneal neovascularization after injury Inhibition of post-injury pyroptosis could be an effective therapy to promote corneal wound healing., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

The structural integrity of cornea depends on properties of its extracellular matrix, mainly a mixture of collagen fibers and soluble proteoglycans (PGs). PGs are macromolecules of negatively charged sulphated glycosaminoglycans (GAGs) covalently attached to a protein core. GAGs appear as bridges between adjacent collagen fibers and could facilitate force transfer between them. Furthermore, GAGs are responsible for corneal hydration by attracting and maintaining water molecules into the extracellular matrix. Based on these observations, GAGs are expected to be essential for biomechanical properties of cornea. The primary objective of the present study was to determine the effects of GAGs on shear properties of cornea. For this purpose, GAGs were enzymatically removed from porcine corneal disks by keratanase II enzyme. After confirming the successful removal of GAGs by histochemical methods, torsional rheometry was performed to characterize the shear stiffness of GAG-depleted samples as a function of axial strain. It was found that the shear modulus of all samples was a function of applied shear strain and compressive strain. Beyond the range of linear viscoelastic response, the average complex shear modulus decreased with increasing the shear strain. Increasing the axial strain from 0% to 40% significantly increased the average complex shear modulus of corneal disks in all groups. Finally, the enzyme treatment with keratanase II enzyme significantly decreased the shear stiffness. The experimental measurements were discussed in terms of microstructural and compositional properties of corneal extracellular matrix and it was concluded that GAGs play a significant role in defining shear properties of cornea., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Keratoconus (KC) is a progressive corneal ectatic disorder with a high prevalence among Asians. This study aimed to explore the differential gene expression patterns in Han Chinese patients with KC, focusing on mRNAs and long noncoding RNAs (lncRNAs), to provide insights into the pathogenesis of the disease. Corneal tissues from KC patients and healthy controls were collected, and RNA sequencing was performed to profile mRNA and lncRNA expression. A total of 1973 differentially expressed mRNAs (DEGs) and 386 differentially expressed lncRNAs (DELs) were identified in KC-affected corneas. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed significant enrichment in pathways related to ECM modulation, PI3K-Akt pathway and calcium signaling pathway. Furthermore, protein-protein interaction (PPI) network highlighted hub genes involved in ECM remodeling and inflammatory responses. Co-expression analysis of lncRNAs and mRNAs further prioritized 13 DELs linked to these hub genes. RT-qPCR validation confirmed the differential expression of select candidates. A meta-analysis integrating seven datasets from diverse ethnic backgrounds was performed and it suggested ethnic-specific differences in gene expression patterns. This study sheds new light on the molecular mechanisms underlying KC in the Han Chinese population, pinpointing potential therapeutic targets. It also emphasizes the critical role of ethnic-specific gene expression patterns in KC research, highlighting a need for tailored approaches in disease management and treatment., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Keratoconus, a progressive corneal disorder characterized by the thinning and conical protrusion of the cornea because of collagen degradation, poses significant challenges to both clinicians and researchers. Most successful animal models of keratoconus are based on genetic mutations and knock-outs in mice and rats that hinder normal corneal stromal architecture, thickness, or strength. While mice and rat models are suitable to study the molecular mechanism and physiological changes to the cornea, they are not suitable for experimental research; especially for surgical interventions like: deep anterior lamellar keratoplasty (DALK), stromal lenticule addition keratoplasty, and other advanced therapies. This review article comprehensively examines recent advancements in experimental models for keratoconus, focusing on their potential for translational research and the challenges ahead. It explores the historical context of experimental models, focusing on animal-based models, mainly rabbits in particular. These advancements enable researchers to mimic the biomechanical and biochemical alterations observed in keratoconic corneas. While these models offer valuable insights into disease mechanisms and treatment development, several challenges remain in transforming experimental findings into clinical applications., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study introduces a novel approach utilizing a temporary drug-eluting hydrogel corneal patch to prevent neovascularization, alongside a numerical predictive tool for assessing the release and transport kinetics of bevacizumab (BVZ) after the keratoplasty. A key focus was investigating the impact of tear film clearance on the release kinetics and drug transport from the designed corneal patch. The proposed tear drug clearance model incorporates the physiological mechanism of lacrimal flow (tear turnover), distinguishing itself from previous models. Validation against experimental data confirms the model's robustness, despite limitations such as a 2D axisymmetrical framework and omission of blink frequency and saccadic eye movements potential effects. Analysis highlights the significant influence of lacrimal flow on ocular drug transport, with the corneal patch extending BVZ residence time compared to topical administration. This research sets the stage for exploring multi-layer drug-eluting corneal patches as a promising therapeutic strategy in ocular health., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, a novel customized corneal cross-linking (CXL) treatment is explored that utilizes microneedles (MNs) for targeted riboflavin (RF) administration prior to the CXL procedure. Unlike the conventional "one-size-fits-all" approach, this protocol offers an option for more precise and efficacious treatment. To simulate a customized corneal crosslinking technique, four distinct microneedle (MN) molds designs, including circular, semi-circular, annular and butterfly shaped, are crafted for loading an optimized RF-hyaluronic acid solution and for the subsequent fabrication of MN arrays with varying morphologies. These MNs can gently puncture the corneal epithelium while preserving the integrity of the underlying stromal layer. Following the application of these microneedles, RF solution is replenished to enhance the RF content within the stroma through the punctures created by the MNs, resulting in exceptional customized corneal cross-linking effects that are comparable to the conventional epi-off CXL protocol. Additionally, it flattened the corneal curvature within the treated zone and facilitated rapid postoperative recovery of corneal tissue. These findings suggest that the integration of customized microneedle RF delivery with corneal crosslinking technology represents a potential novel treatment modality, holding promise for the tailored treatment of corneal pathologies, and offering a more precise and efficient alternative to traditional methods., (© 2024 Wiley‐VCH GmbH.)

The ocular administration of lipophilic and labile drugs such as epalrestat, an aldose reductase inhibitor with potential for diabetic retinopathy treatment, demands the development of topical delivery systems capable of providing sufficient ocular bioavailability. The aim of this work was to develop non-aqueous oleogels based on soybean oil and gelators from natural and sustainable sources (ethyl cellulose, beeswax and cocoa butter) and to assess their reproducibility, safety and efficiency in epalrestat release and permeation both ex vivo and in vivo. Binary combinations of gelators at 10% w/w resulted in solid oleogels (oleorods), while single gelator oleogels at 5% w/w remained liquid at room temperature, with most of the oleogels displaying shear thinning behavior. The oleorods released up to 4 µg epalrestat per mg of oleorod in a sustained or burst pattern depending on the gelator (approx. 10% dose in 24 h). The HET-CAM assay indicated that oleogel formulations did not induce ocular irritation and were safe for topical ocular administration. Corneal and scleral ex vivo assays evidenced the permeation of epalrestat from the oleorods up to 4 and 2.5 µg/cm 2 after six hours, respectively. Finally, the capacity of the developed oleogels to sustain release and provide significant amounts of epalrestat to the ocular tissues was demonstrated in vivo against aqueous-based niosomes and micelles formulations loaded with the same drug concentration. Overall, the gathered information provides valuable insights into the development of oleogels for ocular drug delivery, emphasizing their safety and controlled release capabilities, which have implications for the treatment of diabetic neuropathy and other ocular conditions., (© 2024. The Author(s).)

Cigarette smoke (CS) is an important indoor air pollutant associated with an increased risk of ocular surface disease. As the eye's outermost layer, the cornea is highly sensitive to air pollutants like CS. However, the specific mechanisms linking CS exposure to corneal dysfunction have not been fully elucidated. In the present study, we found that CS exposure damages corneal epithelial cells, accompanied by increased iron (Fe 2+ ) levels and lipid peroxidation, both hallmarks of ferroptosis. Ferroptosis inhibitors, including Ferrostatin-1 (Fer-1) and Deferoxamine mesylate (DFO), protect against CS-induced cell damage. To understand the underlying mechanisms, we investigated how CS affects iron and lipid metabolism. Our results showed that CS could upregulate intracellular iron levels by increasing TFRC expression and promote lipid peroxidation by increasing ACSL4 expression. Silencing ACSL4 or TFRC expression prevented CS-induced ferroptosis. Furthermore, we found that the upregulation of TFRC and ACSL4 was driven by increased YAP transcription. Pharmacological or genetic inhibition of YAP effectively prevented corneal epithelial cell ferroptosis under CS stimulation. Additionally, our results suggest that CS exposure could increase O-GlcNAc transferase activity, leading to YAP O-GlcNAcylation. This glycosylation of YAP interfered with its K48-linked ubiquitination, resulting in YAP stabilization. Collectively, we found that CS exposure induces corneal epithelial cell ferroptosis via the YAP O-GlcNAcylation, and provide evidence that CS exposure is a strong risk factor for ocular surface disease., 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 Ltd. All rights reserved.)

Microplastics (MPs) are ubiquitously dispersed in the environment, and undergoing the process of oxidation that alters their physical and chemical properties. Eyes, which directly interface with the external milieu, inevitably encounter MPs. Nonetheless, the ophthalmic toxicity of MPs towards organisms remains unclear. In this study, primary mouse corneal epithelial cells (MCECs), C57BL/6 mice, and CX3Crl GFP/+ mice were utilized to evaluate the toxicity and differences between oxidized low-density polyethylene MPs (modified-MPs) and low-density polyethylene MPs (virgin-MPs) on eyes. The results manifested that virgin-MPs and modified-MPs could be endocytosed by primary MCECs, resulting in a range of cellular damage. Furthermore, they could diminish tear secretion, increase intraocular pressure, and could be internalized into cornea and retina in mice, instigating a series of detrimental reactions. Importantly, modified-MPs exhibited heightened toxicity towards mouse eyes, seemingly due to oxidation enhances the interaction between virgin-MPs/modified-MPs and tissues/cells, and leading to the release of toxic substances increased. In conclusion, our discoveries demonstrate that oxidation exacerbates the harm of virgin-MPs to eyes, and are of great significance for evaluating the risk of MPs to ocular health., 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 Ltd. All rights reserved.)

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.)

Purpose: Osteopontin (OPN) is a glycosylated, secreted phosphoprotein known to be elevated in both human and mouse retinas during various stages of diabetic retinopathy. However, its specific roles in modulating ocular surface dynamics and immune responses in diabetes remain unexplored. This study aims to investigate the role of OPN in the development of ocular surface disease (OSD) in type 2 diabetic (T2D) mice., Methods: Three- to four-week-old C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice were fed a high-fat diet (HFD) and were rendered diabetic by streptozotocin (STZ; 40 mg/kg body weight) in citrate buffer (vehicle); non-diabetic controls were injected with vehicle alone. Diabetes was confirmed if blood glucose levels were >200 mg/dL, measured 1-2 weeks post-STZ injection. Control, age- and sex-matched db/db diabetic mice fed a standard chow diet were also included in this study. Ocular surface inflammation was assessed using ELISA to quantify inflammatory cytokine proteins and wheat germ agglutinin (WGA) staining was utilized to highlight corneal surface irregularities. Clinical signs were evaluated by corneal fluorescein staining, tear production measurements, and tear sodium (Na+) concentration assessments. These evaluations were conducted 4, 6, 8 and 16-weeks post-diabetes onset in WT and OPN-/- mice and were compared to those obtained in non-diabetic controls. Statistical analysis was performed using a two-way ANOVA, with significance set at P < 0.05., Results: Both WT and OPN-/- mice developed T2D within 4 and 8 weeks, respectively, following HFD + STZ treatment. Corneal OPN levels in WT diabetic mice increased ~2-fold at 2 weeks and ~4-fold at 16 weeks compared to non-diabetic controls, with similar elevations observed in their tear fluid. Diabetic db/db mice also exhibited elevated OPN levels in the blood and ocular surface, which persisted as diabetes progressed. Enhanced fluorescein staining, indicating corneal irregularities, appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. Additionally, WGA staining showed a significant reduction in fluorescence intensity in WT mice treated with HFD and STZ, confirming corneal surface irregularities that were delayed in OPN-/- mice. Elevated tear sodium concentration was observed in both WT and OPN-/- diabetic mice without affecting tear production rates. Notably, OPN levels increased early, at week 2, following HFD and STZ treatment, preceding changes in interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9). Upregulation of IL-6 became apparent at 6 weeks in WT mice and was delayed until 10 weeks in OPN-/- mice post-T2D induction., Conclusions: Elevated OPN levels were detected early post-T2D induction in diabetic WT and db/db mice corneas without initial subclinical changes. This early increase in OPN precedes other proinflammatory cytokines associated with eventual ocular surface inflammation as diabetes progresses. Persistence of OPN also correlated with clinical signs such as increased corneal surface irregularities and elevated tear Na+ concentration. Future research will explore OPN's role as a biomarker in ocular surface disease (OSD), including dry eye disease (DED), and investigate its impact on inflammatory processes and other mechanistic pathways in diabetic ocular complications., Competing Interests: No Competing Interests., (Copyright: © 2024 Datta 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.)

Diabetic keratopathy, characterized by corneal structural changes, is a common complication of diabetes mellitus (DM). Docosahexaenoic acid (DHA), an omega-3 fatty acid, has shown potential therapeutic benefits in various diabetic complications. This study aimed to investigate the protective effect of DHA on corneal tissue in streptozotocin (STZ)-induced type 2 DM in rats. Forty male Sprague-Dawley rats were randomly assigned to four groups (n = 10 per group): Control, DHA, DM, and DM + DHA. The DHA group received DHA by oral gavage at a dose of 100 mg/kg daily for 10 days. In the DM group, diabetes was induced by a single intraperitoneal injection of STZ at 50 mg/kg. Confirmation of diabetes induction was based on monitoring fasting blood glucose levels on the third day post-injection. The DM + DHA group underwent the same diabetes induction protocol with STZ and received DHA at 100 mg/kg daily via oral gavage for 10 consecutive days. Corneal tissue samples were collected at the end of the study period for histopathological, immunohistochemical, qRT-PCR, and ELISA analyses. Histopathological analysis showed significant edema, angiogenesis, and degeneration in the DM group compared to the control (p < 0.001). DHA treatment significantly mitigated these changes, approaching control levels (p < 0.01). Immunohistochemistry showed increased VEGFR2 and iNOS expression in the DM group, which was significantly reduced in the DM + DHA group (p < 0.01). qRT-PCR results indicated a significant decrease in Bcl-2 expression (p < 0.001) and an increase in ATF-6, IRE1, NF-κB, TNF-α, IL-1β, NLRP3, Bax, and Caspase-3 expressions in the DM group (p < 0.001). ELISA analyses revealed significantly elevated levels of inflammatory markers NF-κB, TNF-α, IL-1β, and IL-6 in the DM group compared to the control (p < 0.001). DHA treatment significantly upregulated Bcl-2 and downregulated apoptotic and inflammatory markers (p < 0.01). DHA demonstrated significant protective effects against STZ-induced corneal damage in diabetic rats by modulating apoptotic and inflammatory pathways. These findings suggest that DHA may be a promising therapeutic agent for preventing diabetic keratopathy., 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.)

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases through increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated parameters important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anaesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm 2 for 5 min. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration on days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity on day 6. The ruthenium group also exhibited significantly reduced corneal (p < 0.01) and limbal neovascularization (p < 0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images confirmed high density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study is unique that demonstrates in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This approach can prevent toxicity caused by UV-A light and can be an immediate alternative compared to the existing crosslinking procedures that have side effects and clinical risks for the patients., (© 2024. The Author(s).)

This study aims to investigate the effect of the selective MT2 receptor agonist, IIK7, on corneal autophagy and apoptosis, aiming to reduce corneal epithelial damage and inflammation from blue light exposure in mice. Eight-week-old C57BL/6 mice were divided into BL-exposed (BL) and BL-exposed with IIK7 treatment (BL + IIK7 group). Mice underwent blue light exposure (410 nm, 100 J) twice daily with assessments at baseline and on days 3, 7, and 14. Corneal samples were analyzed for MT2 receptor expression, autophagy markers (LC3-II and p62), and apoptosis indicators (BAX expression and TUNEL assay). Then, mice were assigned to normal control, BL, and BL + IIK7. Ocular surface parameters, including corneal fluorescein staining scores, tear volume, and tear film break-up time, were evaluated on days 7 and 14. On day 14, reactive oxygen species (ROS) levels and CD4 + IFN-γ + T cells percentages were measured. The BL group exhibited higher LC3-II and p62 expression, while the BL + IIK7 group showed reduced expression ( p < 0.05). The TUNEL assay showed reduced apoptosis in the BL + IIK7 group compared to the BL group. ROS levels were lower in the BL + IIK7 group. The BL + IIK7 group showed improved ocular surface parameters, including decreased corneal fluorescein staining and increased tear volume. The percentages of CD4 + IFN-γ + T cells indicated reduced inflammatory responses in the BL + IIK7 group. The MT2 receptor agonist IIK7 regulates corneal autophagy and apoptosis, reducing corneal epithelial damage and inflammation from blue light exposure.

The cornea is a vital component of the visual system, and its integrity is crucial for optimal vision. Damage to the cornea resulting from trauma, infection, or disease can lead to blindness. Corneal regeneration using mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) offers a promising alternative to corneal transplantation. MSCs are multipotent stromal cells that can differentiate into various cell types, including corneal cells. They can also secrete a variety of anti-inflammatory cytokines and several growth factors, promoting wound healing and tissue reconstruction. This review summarizes the current understanding of the molecular and cellular mechanisms by which MSCs and MSC-EVs contribute to corneal regeneration. It discusses the potential of MSCs and MSC-EV for treating various corneal diseases, including corneal epithelial defects, dry eye disease, and keratoconus. The review also highlights finalized human clinical trials investigating the safety and efficacy of MSC-based therapy in corneal regeneration. The therapeutic potential of MSCs and MSC-EVs for corneal regeneration is promising; however, further research is needed to optimize their clinical application.

Introduction: Diabetic retinopathy (DR) is a microvascular complication of diabetes in which neurodegeneration has been recently identified as a driving force. In the last years, mesenchymal stromal cells (MSCs) and neurotrophins like Nerve Growth Factor (NGF), have garnered significant attention as innovative therapeutic approaches targeting DR-associated neurodegeneration. However, delivering neurotrophic factors directly in the eye remains a challenge. Hence, this study evaluated the effects of MSCs from human amniotic fluids (hAFSCs) and recombinant human NGF (rhNGF) delivered by human corneal lenticule (hCL) on a high glucose (HG) induced ex vivo model simulating the molecular mechanisms driving DR., Methods: Porcine neuroretinal explants exposed to HG (25 mM for four days) were used to mimic DR ex vivo . hCLs collected from donors undergoing refractive surgery were decellularized using 0.1% sodium dodecyl sulfate and then bioengineered with hAFSCs, microparticles loaded with rhNGF (rhNGF-PLGA-MPs), or both simultaneously. Immunofluorescence (IF) and scanning electron microscopy (SEM) analyses were performed to confirm the hCLs bioengineering process. To assess the effects of hAFSCs and rhNGF, bioengineered hCLs were co-cultured with HG-treated neuroretinal explants and following four days RT-PCR and cytokine array experiments for inflammatory, oxidative, apoptotic, angiogenic and retinal cells markers were performed., Results: Data revealed that HG-treated neuroretinal explants exhibit a characteristic DR-phenotype, including increased level of NF-kB, NOS2, NRF2 GFAP, VEGFA, Bax/Bcl2 ratio and decreased expression of TUBB3 and Rho. Then, the feasibility to bioengineer decellularized hCLs with hAFSCs and rhNGF was demonstrated. Interestingly, co-culturing hAFSCs- and rhNGF- bioengineered hCLs with HG-treated neuroretinal explants for four days significantly reduced the expression of inflammatory, oxidative, apoptotic, angiogenic and increased retinal markers., Conclusion: Overall, we found for the first time that hAFSCs and rhNGF were able to modulate the molecular mechanisms involved in DR and that bioengineered hCLs represents a promising ocular drug delivery system of hAFSCs and rhNGF for eye diseases treatment. In addition, results demonstrated that porcine neuroretinal explants treated with HG is a useful model to reproduce ex vivo the DR pathophysiology., Competing Interests: The authors ND, GC, MA, and TR are employed by Dompé Farmaceutici SpA. ND, MA, MN, AP, and DM are authors of European Patent n° 20179055.7-1109, 09/06/2020, “New Drug Delivery System for Ophtalmic Use”. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pelusi, Hurst, Detta, Pipino, Lamolinara, Conte, Mastropasqua, Allegretti, Di Pietrantonio, Romeo, El Zarif, Nubile, Guerricchio, Bollini, Pandolfi, Schnichels and Mandatori.)

Keratoconus (KC) is a corneal thinning dystrophy that leads to visual impairment. While the cause of KC remains poorly understood, changes in sex hormone levels have been correlated with KC development. This study investigated circulating gonadotropin-releasing hormone (GnRH) in control and KC subjects to determine if this master hormone regulator is linked to the KC pathology. Plasma and saliva were collected from KC subjects (n = 227 and n = 274, respectively) and non-KC controls (n = 58 and n = 101, respectively), in concert with patient demographics and clinical features. GnRH levels in both plasma and saliva were significantly lower in KC subjects compared to controls. This finding was retained in plasma when subjects were stratified based on age, sex, and KC severity. Control and KC corneal fibroblasts (HKCs) stimulated with recombinant GnRH protein in vitro revealed significantly increased luteinizing hormone receptor by HKCs and reduced expression of α-smooth muscle actin with treatment suggesting that GnRH may modulate hormonal and fibrotic responses in the KC corneal stroma. Further studies are needed to reveal the role of the hypothalamic-pituitary-gonadal axis in the onset and progression of KC and to explore this pathway as a novel therapeutic target.

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.)

Corneal ulcers, whether melting or indolent, are common in humans and companion animals. Treatment involves local administration of antibiotic eye drops and corneal healing drugs. Compared to traditional treatments for ulcerative keratitis, herbal medicines offer unique advantages, such as potent anti-inflammatory effects and inhibition of proinflammatory cytokines. Curcumin, extracted from the Curcuma Longa plant, possesses extensive pharmacological properties, such as anti-inflammatory, anti-cancer, and antioxidant properties, and is used in various medicines. In this study, we developed a novel ophthalmic drop hydrogel using a formulation of Curcumin NPs encapsulated with β-cyclodextrin and hyaluronic acid, to accelerate corneal healing and improve the quality of healed structures. The formation of Curcumin NPs into Hyaluronic acid-based hydrogels was characterized by zeta, FTIR, and scanning electron microscope (SEM) analyses. A total of 25 healthy male New Zealand Albino rabbits were experimentally induced with ulcerative keratitis and treated individually with topical medication. Rabbits were divided into five groups. Fluorescein dye staining, corneal clarity score, Schirmer tear test, proinflammatory cytokine measurement, and pathologic factors assessments were used to evaluate the optimised Curcumin NPs with β-cyclodextrin in Hyaluronic acid hydrogel. Our results demonstrated that the optimized Curcumin NPs with β-cyclodextrin in hyaluronic acid hydrogel significantly reduced the frequency of medication administration compared to conventional therapies, enhancing the quality of healed structures and effectively treating ulcerative keratitis. All findings in this study provide new insight into designing and fabricating novel ophthalmic medicine for ulcerative keratitis for topical usage., (© 2024. The Author(s).)

Purpose: This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining., Methods: Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution., Results: We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea., Conclusions: This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.

The cornea protects the interior of the eye from external agents such as bacteria, viruses, and debris. Synthetic glucocorticoids are widely prescribed in the treatment of ocular infections and disorders. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR); however, the molecular and physiological functions of GR signaling in the cornea are poorly understood. This study found that treatment of mice with glucocorticoid eye drops led to a profound regulation of the corneal transcriptome. These glucocorticoid-regulated genes were associated with multiple biological functions, including the immune response. To understand the direct role of GR signaling in the cornea, mice with conditional knockout of GRs in the corneal epithelium were generated. Mice lacking corneal GRs exhibited microphthalmia, loss of pupils, a deformed and opaque lens, and mislocalization of key structural proteins within the corneal epithelial layers. Global transcriptomic approaches revealed that loss of GR signaling in the cornea also resulted in the dysregulation of a large cohort of genes strongly associated with an enhanced inflammatory response. Finally, corneal GR signaling was required for preventing neovascularization of blood and lymphatic vessels and thereby immune cell infiltration of the cornea. These results reveal that corneal GR signaling plays a critical role in ocular development and in maintaining the homeostasis of the eye., Competing Interests: Disclosure Statement None declared., (Published by Elsevier Inc.)

Photobiomodulation (PBM) has emerged as a potentially effective therapeutic approach to modulate cellular functions. This study aimed to examine the impact of PBM on reactive oxygen species (ROS), lipid peroxidation, and glutathione-related antioxidant defense systems in rabbit eye tissues. A polychromatic light source with an intensity of 2.6 J/cm 2 /min was used for PBM treatment in New Zealand White rabbits for 12 min. The PBM group (n = 8) received treatments every 2 days for a total of 12 sessions, whereas the control group (n = 8) did not undergo any PBM light exposure during the same period. The application of PBM significantly elevated ROS-mediated glutathione levels, along with increased activities of glutathione peroxidase and reductase, particularly in corneal tissue (p ≤ 0.05). In conclusion, PBM treatment effectively enhances antioxidant defense mechanisms in the eye, particularly in corneal tissue, suggesting its potential as a therapeutic strategy for managing oxidative stress-related ocular conditions., (© 2024 The Author(s). Journal of Biophotonics published by Wiley‐VCH GmbH.)

Itraconazole (ITZ) is one of the broad-spectrum antifungal agents for treating fungal keratitis. In clinical use, ITZ has problems related to its poor solubility in water, which results in low bioavailability when administered orally. To resolve the issue, we formulated ITZ into the inclusion complex (ITZ-IC) system using β-cyclodextrin (β-CD), which can potentially increase the solubility and bioavailability of ITZ. The molecular docking study has confirmed that the binding energy of ITZ with the β-CD was -5.0 kcal/mol, indicating a stable conformation of the prepared inclusion complex. Moreover, this system demonstrated that the inclusion complex could significantly increase the solubility of ITZ up to 4-fold compared to the pure drug. Furthermore, an ocular drug delivery system was developed through dissolving microneedle (DMN) using polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) as polymeric substances. The evaluation results of DMN inclusion complexes (ITZ-IC-DMN) showed excellent mechanical strength and insertion ability. In addition, ITZ-IC-DMN can dissolve rapidly upon application. The ex vivo permeation study revealed that 75.71% (equivalent to 3.79 ± 0.21 mg) of ITZ was permeated through the porcine cornea after 24 h. Essentially, ITZ-IC-DMN exhibited no signs of irritation in the HET-CAM study, indicating its safety for application. In conclusion, this study has successfully developed an inclusion complex formulation containing ITZ using β-CD in the DMN system. This approach holds promise for enhancing the solubility and bioavailability of ITZ through ocular administration.

The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self-renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re-epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non-human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The mammalian cornea is decorated with stem cells bestowed with the life-long task of renewing the epithelium, provided they remain healthy, functional, and in sufficient numbers. If not, a debilitating disease known as limbal stem cell deficiency (LSCD) can develop causing blindness. Decades after the first stem cell (SC) therapy is devised to treat this condition, patients continue to suffer unacceptable failures. During this time, improvements to therapeutics have included identifying better markers to isolate robust SC populations and nurturing them on crudely modified biological or biomaterial scaffolds including human amniotic membrane, fibrin, and contact lenses, prior to their delivery. Researchers are now gathering information about the biomolecular and biomechanical properties of the corneal SC niche to decipher what biological and/or synthetic materials can be incorporated into these carriers. Advances in biomedical engineering including electrospinning and 3D bioprinting with surface functionalization and micropatterning, and self-assembly models, have generated a wealth of biocompatible, biodegradable, integrating scaffolds to choose from, some of which are being tested for their SC delivery capacity in the hope of improving clinical outcomes for patients with LSCD., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Purpose: Inter-eye variability is a recognized characteristic of Dry Eye Disease (DED) and has been proposed as a diagnostic indicator in clinical practice. This study aimed to assess the diagnostic potential of the absolute difference between eyes in three key diagnostic tests recommended by the Tear Film and Ocular Surface Society Dry Eye Workshop II (TFOS DEWS II) Diagnostic Methodology report: tear film osmolarity, Fluorescein Break-Up Time (FBUT), and ocular surface staining., Methods: A total of 180 participants were included in a cross-sectional study. Before a dry eye examination, participants completed an online self-administered OSDI questionnaire. The TFOS DEWS II diagnostic criteria for DED assessment were used: along with OSDI, osmolarity, FBUT and ocular surface staining were measured in all participants in both eyes following standardized methodology. Based on signs and symptoms, participants were diagnosed as having No DED or DED. After diagnosis, the parameters were computed as the right and left eyes' absolute inter-eye difference (|OD-OS|)., Results: Receiver Operating Characteristics analyses for computed parameters were used based on the previous diagnosis. ROC analyses showed that Osmolarity|OD-OS| have a diagnostic capability to differentiate between No DED and DED participants with a cut-off value of 9.5 mOsm/L (AUC = 0.745 ± 0.052, p  < 0.003), whereas FBUT|OD-OS| and Corneal Stainning|OD-OS| have not (AUC, both p  ≥ 0.160)., Conclusion: The present study found that the Osmolarity|OD-OS| parameter could be used as a diagnostic indicator for DED assessment, while the FBUT|OD-OS| and the Corneal Staining|OD-OS| parameters do not have this capability.

Dry eye disease (DED) is a common ocular condition affecting a significant portion of the global population, yet effective treatment options remain elusive. This study investigates the therapeutic potential of M2 macrophage-derived extracellular vesicles (M2-EVs) in a mouse model of DED. The DED model was established using 0.2% benzalkonium chloride (BAC) eye drops, applied twice daily for a week. Post induction, the mice were categorized into 5 groups: PBS, Sodium Hyaluronate (HA, 0.1%), Fluoromethalone (FM, 0.1%), M0-EVs, and M2-EVs. The efficacy of M2-EVs was assessed through tear production, corneal fluorescein staining and HE staining. RNA sequencing (RNA-seq) was employed to investigate the mechanisms underlying the therapeutic effects of M2-EVs in DED. Notably, the M2-EVs treated group exhibited the highest tear secretion, indicating improved tear film stability and reduced corneal surface damage. Histological analysis revealed better corneal structure organization in the M2-EVs group, suggesting enhanced ocular surface repair and corneal preservation. Furthermore, M2-EVs treatment significantly decreased pro-inflammatory cytokine levels and showed unique enrichment of genes related to retinal development. These findings suggest that M2-EVs could serve as a promising noninvasive therapeutic approach for human DED, targeting ocular surface inflammation., Competing Interests: Declaration of competing interest No., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacterial biofilm formation protects bacteria from antibiotics and the immune system, excessive inflammation further complicates treatment. Here, iron-based metal-organic framework (MIL-101)-loaded riboflavin nanoparticles are designed for the therapeutic challenge of biofilm infection and hyperinflammation in bacterial keratitis. Specifically, MIL-101 produces a thermal effect under exogenous near-infrared light irradiation, which synergizes with ferroptosis-like bacterial death induced by iron ions to exert an effective biofilm infection eradication effect. On the other hand, the disintegration of MIL-101 sustains the release of riboflavin, which inhibits the pro-inflammatory response of macrophage over-activation by modulating their phenotypic switch. In addition, to solve the problems of short residence time, poor permeability, and low bioavailability of corneal medication, the MR@MN microneedle patch is further prepared by loading nanoparticles into SilMA hydrogel, which ultimately achieves painless, transepithelial, and highly efficient drug delivery. In vivo and ex vivo experiments demonstrate the effectiveness of this approach in eliminating bacterial infection and promoting corneal healing. Therefore, the MRMN patch, acting as an ocular drug delivery system with the ability of rapid corneal healing, promises a cost-effective solution for the treatment of bacterial keratitis, which may also lead to a new approach for treating bacterial keratitis in clinics., (© 2024 Wiley‐VCH GmbH.)

Purpose: Corneal fibroblasts are involved in the wound healing of the cornea with proliferation, migration, and differentiation processes. Coenzyme Q10 (CoQ10) and vitamin E can enhance corneal wound healing when applied after a corneal lesion as an eye drop. Thus, this study was performed to determine the potential efficiency of a CoQ10 ophthalmical solution containing a CoQ10 and vitamin E D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-derived formulation in human corneal fibroblasts (HCFs) in vitro . Methods: Primary HCFs were obtained from cadaveric corneal tissue, and cell viability was determined using MTT assay at 24 and 72 h. Cell migration was evaluated using an in vitro wound healing assay, and mRNA expressions of collagen type I (COL-I), collagen type III (COL-III), lumican, hyaluronan, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitors of MMP (TIMP)-1, TIMP-2, interleukin (IL)-1β, IL-6, IL-8, and IL-10 were assessed using reverse transcription polymerase chain reaction at 24 and 72 h. Results: At various concentrations of CoQ10 ophthalmical solution (CoQ10-os), cell viability and wound healing rates of HCFs increased compared with the control group. The expressions of COL-I , COL-III , lumican, and hyaluronan were increased by CoQ10-os, whereas those of MMP-1 , MMP-2 , MMP-9 , TIMP-1 , TIMP-2 , and TIMP-3 were not affected by CoQ10-os at 24 and 72 h. In treating HCFs with a CoQ10-os medium, IL-1β , IL-6 , and IL-8 decreased, whereas IL-10 was significantly increased in a time- and dose-dependent manner. Conclusions: The findings indicate that CoQ10 and vitamin E-TPGS are potent regulators of the bioactivity of HCFs, thus supporting their potential application as ophthalmical solutions in therapies aimed at the fast regeneration of damaged cornea tissues.

Keratomycosis, caused by pathogenic fungi, is an intractable blinding eye disease. Corneal penetration is an essential requirement for conventional antifungal medications to address keratomycosis. Due to the distinctive anatomical and physiological structure of the cornea, the therapeutic efficacy is hampered by the inadequate penetration capacity. Despite the emergence of diverse antifungal drug delivery systems and advanced antifungal nanomaterials, it has remained challenging to achieve corneal penetration over the past decade. This study fabricates a penetrative ionic organic molecular cage-based nanozyme (OMCzyme) for treating keratomycosis. The synthesis of OMCzyme involved two steps. Initially, the ionic OMC is synthesized by a [2+3] cycloimination reaction of triformylphloroglucinol and 2,3-diaminopropionic acid. Subsequently, OMCzyme is fabricated by coordination of Fe 2 ⁺ with carboxyl anions and phenolic hydroxyls in the organic cage, and further deposition of silver nanoparticles on the surface of OMC-Fe complex. The as-prepared OMCzyme demonstrates excellent water dispersion, peroxidase-like activity, in vitro and in vivo biocompatibility, and corneal penetration. Notably, the nanozyme displays targeted antifungal activity, effectively combating Fusarium solani with negligible cytotoxicity toward human corneal epithelial cells. The hybrid mimic is further demonstrated to be effective in treating keratomycosis in mice, indicating the potential of OMCzyme for curing fungal infectious diseases., (© 2024 Wiley‐VCH GmbH.)

Envisaging to improve the evaluation of ophthalmic drug products while minimizing the need for animal testing, our group developed the OphthalMimic device, a 3D-printed device that incorporates an artificial lacrimal flow, a cul-de-sac area, a moving eyelid, and a surface that interacts effectively with ophthalmic formulations, thereby providing a close representation of human ocular conditions. An important application of such a device would be its use as a platform for dissolution/release tests that closely mimic in vivo conditions. However, the surface that artificially simulates the cornea should have a higher resistance (10 min) than the previously described polymeric films (5 min). For this key assay upgrade, we describe the process of obtaining and thoroughly characterizing a hydrogel-based hybrid membrane to be used as a platform base to simulate the cornea artificially. Also, the OphthalMimic device suffered design improvements to fit the new membrane and incorporate the moving eyelid. The results confirmed the successful synthesis of the hydrogel components. The membrane's water content (86.25 ± 0.35 %) closely mirrored the human cornea (72 to 85 %). Furthermore, morphological analysis supported the membrane's comparability to the natural cornea. Finally, the performance of different formulations was analysed, demonstrating that the device could differentiate their drainage profile through the viscosity of PLX 14 (79 ± 5 %), PLX 16 (72 ± 4 %), and PLX 20 (57 ± 14 %), and mucoadhesion of PLXCS0.5 (69 ± 1 %), PLX16CS1.0 (65 ± 3 %), PLX16CS1.25 (67 ± 3 %), and the solution (97 ± 8 %). In conclusion, using the hydrogel-based hybrid membrane in the OphthalMimic device represents a significant advancement in the field of ophthalmic drug evaluation, providing a valuable platform for dissolution/release tests. Such a platform aligns with the ethical mandate to reduce animal testing and promises to accelerate the development of safer and more effective ophthalmic drugs., 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.)

The cornea serves as a vital protective barrier for the eye; however, it is prone to injury and damage that can disrupt corneal epithelium and nerves, triggering inflammation. Therefore, understanding the biological effects and molecular mechanisms involved in corneal wound healing and identifying drugs targeting these pathways is crucial for researchers in this field. This study aimed to investigate the therapeutic potential of progranulin (PGRN) in treating corneal injuries. Our findings demonstrated that PGRN significantly enhanced corneal wound repair by accelerating corneal re-epithelialization and re-innervation. In vitro experiments with cultured epithelial cells and trigeminal ganglion cells further revealed that PGRN stimulated corneal epithelial cell proliferation and promoted axon growth in trigeminal ganglion cells. Through RNA-sequencing (RNA-seq) analysis and other experimental techniques, we discovered that PGRN exerted its healing effects modulating Wnt signaling pathway, which played a critical role in repairing epithelial cells and promoting axon regeneration in trigeminal neurons. Importantly, our study highlighted the anti-inflammatory properties of PGRN by inhibiting the NF-κB signaling pathway, leading to decreased infiltration of macrophages. In conclusion, our findings underscored the potential of PGRN in facilitating corneal wound healing by promoting corneal epithelial cell proliferation, trigeminal ganglion cell axon regeneration, and suppressing ocular inflammation. These results suggest that PGRN could potentially expedite the healing process and improve visual outcomes in patients with corneal injuries., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Corneal wound healing in diabetic patients is usually delayed and accompanied by excessive inflammation. However, the underlying cellular and molecular mechanisms remain poorly understood. Here, we found that somatostatin (SST), an immunosuppressive peptide produced by corneal nerve fibers, was significantly reduced in streptozotocin-induced diabetic mice. In addition, we discovered that topical administration of exogenous SST significantly improved re-epithelialization and nerve regeneration following diabetic corneal epithelial abrasion. Further analysis showed that topical SST significantly reduced the expression of injury inflammation-related genes, inhibited neutrophil infiltration, and shifted macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 in diabetic corneas' healing. Moreover, the application of L-817,818, an agonist of the SST receptor type 5 subtype, significantly reduced the inflammatory response following epithelial injury and markedly improved the process of re-epithelialization and nerve regeneration in mice. Taken together, these data suggest that activation of the SST-SST receptor type 5 pathway significantly ameliorates diabetes-induced abnormalities in corneal wound repair in mice. Targeting this pathway may provide a novel strategy to restore impaired corneal wound closure and nerve regeneration in diabetic patients., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: We aimed to prepare a β-cyclodextrin (β-CD) polymer using radical polymerization with co-monomers, 6-deoxy-6-(2-methacryloyloxyethylsuccinamide)-β-cyclodextrin (CD-MSAm) and N,N,N-trimethyl-N-(2-hydroxy-3-metacryloyloxopropyl)-ammonium chloride (QA) to design cyclodextrins suitable for use in ophthalmology. In addition, we evaluated their solubility and inclusion properties with rebamipide (REB), a poorly soluble drug, and investigated the usefulness of the β-CD polymer and REB (REB@CDQA) combination in treating dry eye., Methods: The β-CD polymer (CD-MSAm-co-QA, CDQA) based on CD-MSAm/QA was prepared via radical polymerization, and the usefulness of REB@CDQA in treating dry eye was evaluated using a rabbit treated with N-acetylcysteine (dry eye model)., Results: The solubility of the CDQA powder was higher than that of the β-CD powder, and 80 nm colloids were observed in the CDQA solution. No corneal toxicity was observed in human corneal epithelial cells or rat corneas treated with 0.2% CDQA solution. The levels of REB dissolved in the CDQA solution were higher than those of the β-CD solution. Moreover, the application of the CDQA solution enhanced REB retention in the cornea and attenuated the transcorneal penetration of REB. In addition, instillation of REB@CDQA enhanced the volume of the lacrimal fluid and normalized the reduced mucin levels in the dry eye model. The extent of tear film breakup was attenuated by REB@CDQA instillation., Conclusion: The CDQA solution enhanced the solubility of REB, and the combination of CDQA and REB enhanced the drug content in the corneal tissue. Moreover, the therapeutic effect on dry eye was higher than that of REB suspensions without CDQA., Competing Interests: Ko Kobayashi, Masaru Matsuda, Norio Iwakiri, and Eiji Harata worked for NOF CORPORATION. The authors declare no other conflicts of interest in this work., (© 2024 Otake et al.)

Topical eye drop approaches to treat ocular inflammation in dry eyes often face limitations such as low efficiency and short duration of drug delivery. Nanofibers serve to overcome the limitation of the short duration of action of topical eye drops used against ocular inflammation in dry eyes. Several attempts to develop suitable nanofibers have been made; however, there is no ideal solution. Here, we developed polycaprolactone (PCL) nanofibers loaded with dexamethasone acetate (DEX), prepared by electrospinning, as a potential ocular drug delivery platform for corneal injury treatment. Thirty-nine Sprague Dawley rats (7 weeks old males) were divided into four treatment groups after alkaline burns of the cornea; negative control (no treatment group); dexamethasone eyedrops (DEX group); PCL fiber (PCL group); dexamethasone loaded PCL (PCL + DEX group). We evaluated therapeutic efficacy of PCL + DEX by examining the epithelial wound healing effect, the extent of corneal opacity and neovascularization. Additionally, various inflammatory factors, including IL-1β, were investigated through immunochemistry, western blot analysis, and quantitative real-time RT-PCR (qRT-PCR). PCL + DEX group showed histologically alleviated signs of corneal inflammation compared with DEX group, which showed a decrease in IL-1β and MMP9 in the corneal stroma. The quantitative expression on day 1 after alkaline burn of pro-inflammatory markers, including IL-1β and IL-6, in the PCL + DEX group was significantly lower than that in the DEX group. Notably, PCL + DEX treatment significantly suppressed neovascularization, and enhanced the anti-inflammatory function of DEX during the acute phase of ocular inflammation. Collectively, these findings suggest that PCL + DEX may be a promising approach to effective drug delivery in corneal burn injuries., (© 2024. The Author(s).)

Oxygen (O 2 ) metabolism plays a critical role in cornea wound healing, regeneration, and homeostasis; however, the underlying spatiotemporal mechanisms are poorly understood. Here we used an optical sensor to profile O 2 flux in intact and wounded corneas of mouse eyes. Intact corneas have unique centrifugal O 2 influx profiles, smallest flux at the cornea center, and highest at the limbus. Following cornea injury, the O 2 influx profile presents three distinct consecutive phases: a "decreasing" phase from 0 to 6 h, a "recovering" phase from 12 to 48 h, and a 'peak' phase from 48 to 72 h, congruent to previously described healing phases. Immediately after wounding, the O 2 influx drops at wound center and wound edge but does not change significantly at the wound side or limbus. Inhibition of reactive oxygen species (ROS) in the decreasing phase significantly reduces O 2 influx, decreases epithelial migration and consequently delays healing. The dynamics of O 2 influx show a positive correlation with cell proliferation at the wound side, with significantly increased proliferation at the peak phase of O 2 influx. This study elucidates the spatiotemporal O 2 dynamics in both intact and wounded rodent cornea and shows the crucial role of O 2 dynamics in regulating cell migration and proliferation through ROS metabolism, ultimately contributing to wound healing. These results demonstrate the usefulness of the micro-optrode in the characterization of spatiotemporal O 2 dynamics. Injury-induced changes in O 2 metabolism and ROS production modulate O 2 dynamics at wound and control cell migration and proliferation, both essential for proper wound healing., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

To assess the impact of glucocorticosteroids with varying potencies on inflammatory mediators in tears and corneal optical density after femtosecond-assisted laser in situ keratomileusis (FS-LASIK). In a prospective study, 110 patients (220 eyes) who underwent FS-LASIK were divided into 2 groups: 55 patients (110 eyes) received dexamethasone, and another 55 patients (110 eyes) received fluorometholone. Visual acuity, intraocular pressure, and corneal optical density were measured before, 1 week, and 1 month after surgery. Tear fluid samples were also collected to assess expression levels of TNF-α, IL-1α, IL-6, and TGF-β1. One week after the procedure, the dexamethasone group exhibited elevated intraocular pressure (IOP) levels (P > .05) and a decreased expression of TNF-α in tears (P < .001) compared to the fluorometholone group. Within the 0 to 2 mm range from the corneal apex, the anterior corneal layer's optical density in the fluorometholone group surpassed that of the dexamethasone group (P < .05). At 1 month post-surgery, the IOP in the fluorometholone group was higher than that in the dexamethasone group (P < .05). In both the 0 to 2 mm and 2 to 6 mm intervals from the corneal apex, the optical density of the anterior corneal layer was significantly higher in the fluorometholone group compared to the dexamethasone group (P < .05). There was no statistically significant difference in visual acuity between the 2 groups at any postoperative time point. Short-term use of potent corticosteroids after FS-LASIK can swiftly address ocular surface inflammation, enhance corneal wound healing, reduce corneal edema, and accelerate the restoration of corneal transparency, in contrast to prolonged use of milder corticosteroids post-surgery., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

The factors that contribute to pain after nerve injury remain incompletely understood. Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) are common surgical techniques to correct refractive errors. After LASIK or PRK, a subset of patients suffers intense and persistent pain, of unknown origin, described by patients as feeling like shards of glass in their eye. Here, we evaluated a TRPV1 variant, p.V527M, found in a 49-y-old woman who developed corneal pain after LASIK and subsequent PRK enhancement, reporting an Ocular Surface Disease Index score of 100. Using patch-clamp and Ca 2+ imaging, we found that the V527M mutation enhances the response to acidic pH. Increasing proton concentration induced a stronger leftward shift in the activation curve of V527M compared to WT, resulting in channel activity of the mutant in acidic pH at more physiological membrane potentials. Finally, comparing the responses to consecutive applications of different agonists, we found in V527M channels a reduced capsaicin-induced desensitization and increased sensitization by the arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). We hypothesize that the increased response in V527M channels to protons and enhanced sensitization by 12-HETE, two inflammatory mediators released in the cornea after tissue damage, may contribute to the pathogenesis of corneal neuralgia after refractive surgery., Competing Interests: Competing interests statement:The authors declare no competing interest.

Genistein (Gen); a naturally occurring isoflavone, acts as a tyrosine kinase inhibitor and efficiently downregulates inflammatory cytokines, which are pivotal in eye inflammation. Also, Gen suffers from sparse ocular bioavailability due to poor solubility. In this work, nanostructured lipid carriers (NLCs) were successfully fabricated by using solid (stearic acid and compritol) and liquid (oleic acid) lipids. The optimized Gen-loaded NLCs showed a nanosize range of 140-246 nm, ≥ 98 % entrapment efficiency, and controlled release over 48 h. The ζ-potential of NLCs was increased from -27.3 mV to 25-27.4 mV due to surface modification with chitosan (CS) or eudragit RS100 (ERS 100). All NLCs showed prominent biocompatibility with enhanced cellular uptake on corneal stromal fibroblasts. Moreover, the different NLCs were incorporated into a mucoadhesive in situ gel. The optimized in situ gel (G9), containing 20 % poloxamers and 0.5 % hydroxyethyl cellulose, exhibited excellent gelling ability within 10.5 s, gelling temperature at 33.1 ± 0.6 ℃, spreadability diameter of 4.73 ± 0.12 cm, shear-thinning behavior, and 20 min ex vivo mucoadhesion time with drug release for 120 h. The in vivo results showed distinguished permeation and distribution potential for ocular delivery. In vivo anti-inflammatory effects after 3 days of treatment with CS-Gen-NLCs/G9 and ERS-Gen-NLCs/G9 revealed a downregulation of interleukin-6 levels in the cornea and retina compared to the untreated group. Our research highlights the promising anti-inflammatory potential of ERS-Gen-NLCs/G9 as an efficient, non-irritant Gen nanodelivery system for managing anterior and posterior ocular inflammation., 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.)

Purpose: Transdifferentiation of corneal fibroblasts to myofibroblasts in the stroma is a central mechanistic event in corneal wound healing. This study sought to characterize genes and pathways influencing transdifferentiation of human corneal fibroblasts (hCSFs) to human corneal myofibroblasts (hCMFs) using RNA sequencing (RNA-seq) to develop comprehensive mechanistic information and identify newer targets for corneal fibrosis management., Methods: Primary hCSFs were derived from donor human corneas. hCMFs were generated by treating primary hCSFs with transforming growth factor β1 (TGFβ1; 5 ng/mL) for 72 hours under serum-free conditions. RNA was extracted using the RNeasy Plus Mini Kit and subjected to RNA-seq analysis after quality control testing. Differential gene expression, pathway enrichment, and protein-protein network analyses were performed using DESeq2, GSEA/PANTHER/Reactome, and Cytoscape/cytoHubba, respectively., Results: RNA-seq analysis of hCMFs and hCSFs identified 3843 differentially expressed genes and transcripts (adjusted P < 0.05). The log(fold change) ≥ ±1.5 filter showed 816 upregulated and 739 downregulated genes between two cell types. Pathway enrichment analysis showed the highest normalized enrichment score for epithelial-to-mesenchymal transition (5.569), followed by mTORC1 signaling (2.949), angiogenesis (2.176), and TGFβ signaling (2.008). Protein-protein interaction network analysis identified the top 20 nodes influencing corneal myofibroblast development. The expression of a novel MXRA5 in corneal stroma and its association with corneal fibrosis was verified by real-time quantitative reverse transcription PCR and immunofluorescence. RNA-seq and gene count files were submitted to the NCBI Gene Expression Omnibus (GSE260476)., Conclusions: This study identified several novel genes involved in myofibroblast development, offering potential targets for developing newer therapeutic strategies for corneal fibrosis.