Conjunctival Goblet Cell Density Research Articles

Characterizing the Robustness of Distinct Clinical Assessments in Identifying Dry Eye Condition of Animal Models

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 Role of IL-6, IL-10, and TNF-α in Ocular GVHD Following Allogeneic Transplantation.

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. 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. 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. Dry eye symptoms related to ocular GVHD, can be partly diagnosed and assessed using various tear cytokine level detection methods.

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

Adverse effects of BAK in glaucoma medications

Benzalkonium chloride (BAK) is a common preservative found in glaucoma medications used to increase shelf‐life, prevent bacterial contamination, and enhance ocular penetration. Its detergent properties kill microorganisms by disrupting cell membranes and causing cell lysis. Unfortunately BAK also has potentially harmful cytotoxic effects, particularly for the ocular surface, with evidence of BAK‐induced apoptosis of conjunctival and corneal epithelial cells, decreased density of conjunctival goblet cells, delayed corneal wound healing and reduced density of corneal nerved. It may also affect deeper ocular structures including the lens and trabecular meshwork, with BAK shown to cause apoptosis of human trabecular meshwork cells in culture. Clinical consequences may include increased risk of ocular surface disease, reduced adherence, and with long‐term use, reduced success of glaucoma surgery.

Purple Corn Extract Improves Dry Eye Symptoms in Models Induced by Desiccating Stress and Extraorbital Lacrimal Gland Excision.

Dry eye disease (DED) occurs when there are not enough tears, and the associated symptoms-burns, itching, and a gritty feeling in the eye-can cause great discomfort. The purpose of this study was to evaluate the therapeutic effect of purple corn extract (PCE) on DED. Pretreatment with PCE prevented desiccation-stress-induced cell damage in human retinal pigment epithelial cells and primary human corneal epithelial cells. Furthermore, PCE reduced the mRNA expression of inflammatory mediators in the induction of desiccation stress. The therapeutic effects of PCE on DED were evaluated in an animal model with induced unilateral excision of the exorbital lacrimal gland. The administration of PCE was effective at recovering tear production, corneal surface irregularity, and conjunctival goblet cell density, as well as at reducing apoptotic cell death in the outer layer of the corneal epithelium. Collectively, PCE improved dry eye symptoms, and, therefore, it could be a potential agent to ameliorate and/or treat DED.

Potential Benefits of Integrin αvβ3 Antagonists in a Mouse Model of Experimental Dry Eye.

The purpose of this study was to extensively evaluate the efficacy of integrin αvβ3 antagonists for the treatment of experimental dry eye (EDE). Vitronectin, an αvβ3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvβ3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. The inhibitory effects of αvβ3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvβ3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. Topical applications of αvβ3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvβ3 may be a novel promising strategy to treat patients with dry eye disease.

Efficacy and safety of platelet-rich plasma and autologous-serum eye drops for dry eye in primary Sjögren’s syndrome: a randomized trial

We compared the efficacy and safety of autologous-serum (AS) and platelet-rich plasma (PRP) eye drops for dry eye (DE) treatment in primary Sjögren’s syndrome (SS). This prospective, randomized, double-blinded clinical study included patients diagnosed with primary SS DE. Thirty-eight participants were randomly assigned to the AS or PRP groups. Corneal and conjunctival staining scores, Schirmer I test, tear film break-up time (TBUT), and ocular surface disease index (OSDI) scores were evaluated at 4 and 12 weeks. Conjunctival impression cytology (CIC) metaplasia grade and goblet cell density grade at 12 weeks were compared with those at baseline. Corneal and conjunctival staining scores and TBUT significantly improved at 4 and 12 weeks in both groups (all p < 0.005). No significant difference between the AS and PRP groups was observed at 4 and 12 weeks. The Schirmer I values, OSDI scores, CIC metaplasia grade, and goblet cell density grade did not significantly change at 4 and 12 weeks in either group. Both AS and PRP eye drops are effective for primary SS DE without a significant difference. Considering that the preparation time of PRP is shorter than that of AS, PRP can be a good alternative treatment for primary SS DE.

Investigation of Conjunctival Goblet Cell and Tear MUC5AC Protein in Patients With Graves' Ophthalmopathy.

The aim of this study was to investigate conjunctival goblet cell density (GCD) and tear mucin-5AC (MUC5AC) protein levels in patients with Graves' ophthalmopathy (GO) and their association with dry eye indicators. A total of 99 patients with GO (54 active, 45 inactive) and 40 healthy controls were recruited. Comprehensive ophthalmic examinations, including the external eye, ocular surface, GCD, and tear MUC5AC ELISA, were performed. The GCD examination was performed in temporal bulbar conjunctiva, including IVCM GCD by in vivo confocal microscopy (IVCM) and filled GCD of cytokeratin-7 and MUC5AC-positive co-immunomarkers by impression cytology. Tear MUC5AC protein was detected using samples extracted from Schirmer strips. The GO group showed a significant decrease in IVCM GCD, filled GCD, and normalized tear MUC5AC protein compared to controls, with the active GO group showing the greatest decrease (all P<0.05). Tear MUC5AC protein levels in GO correlated with those of IVCM GCD (r = 0.40, P<0.001) and filled GCD (r = 0.54, P<0.001, respectively). Higher ocular surface disease index (r = -0.22, P<0.05; r = -0.20, P<0.05; r = -0.21, P<0.05) and lisamine green staining (r = -0.23, P<0.05; r = -0.38, P<0.001; r = -0.42, P<0.001) were associated with lower tear MUC5AC protein levels, IVCM GCD, and filled GCD, respectively, which decreased with increasing clinical activity score (r = -0.24, P<0.05; r = -0.28, P<0.01; r = -0.27, P<0.01) and conjunctival congestion score (r = -0.27, P<0.01; r = -0.33, P<0.001; r = -0.42, P<0.001). The goblet cell count and tear MUC5AC protein in GO eyes were decreased, possibly due to ocular surface inflammation. This study observed the change of tear film mucin in GO patients.

Bacillus-Derived Manganese Superoxide Dismutase Relieves Ocular-Surface Inflammation and Damage by Reducing Oxidative Stress and Apoptosis in Dry Eye.

We hypothesized that antioxidative enzymes supplementation could be a treatment option for dry eye. We investigated the efficacy of oral administration of Bacillus-derived superoxide dismutase (Bd-SOD) in a murine experimental dry eye (EDE). In part I, mice were randomly assigned to normal control, EDE, and mice groups that were treated with oral Bd-SOD after induction of EDE (EDE + Bd-SOD group; four mice in each group). Expression of SOD2, a major antioxidant enzyme with manganese as a cofactor, was assessed by immunofluorescence staining. In part II, mice were divided into seven groups (six mice in each group): normal control, EDE, vehicle-treated, topical 0.05% cyclosporin A (CsA)-treated, and oral Bd-SOD-treated (2.5, 5.0, and 10.0mg/kg Bd-SOD) groups. Tear volume, tear-film break-up time (TBUT), and corneal fluorescein-staining scores (CFS) were measured at zero, five, and 10days after treatment. Tendays after treatment, 2',7'-dichlorodihydrofluorescein diacetate for reactive oxygen species (ROS), enzyme-linked immunosorbent for malondialdehyde, and TUNEL assays for corneal apoptosis, flow cytometry inflammatory T cells, and histological assessment were performed. Compared to the normal control group in part I, the EDE group showed significantly decreased SOD2 expression by immunofluorescence staining. However, the EDE + Bd-SOD group recovered similar to the normal control group. In part II, ROS, malondialdehyde, and corneal apoptosis were decreased in CsA and all Bd-SOD-treated groups. Corneal and conjunctival inflammatory T cells decreased, and conjunctival goblet cell density increased in CsA-treated and Bd-SOD-treated groups. Compared to the CsA-treated group, the 2.5mg/kg Bd-SOD-treated group showed increased TBUT and decreased inflammatory T cells, and the 5.0mg/kg Bd-SOD-treated group showed decreased CFS and increased conjunctival goblet cells. Oral Bd-SOD administration might increase autogenous SOD2 expression in ocular surface tissue in EDE and could be developed as a complementary treatment for DE in the future.

Effects of Cathepsin S Inhibition in the Age-Related Dry Eye Phenotype.

Aged C57BL/6J (B6) mice have increased levels of cathepsin S, and aged cathepsin S (Ctss-/-) knockout mice are resistant to age-related dry eye. This study investigated the effects of cathepsin S inhibition on age-related dry eye disease. Female B6 mice aged 15.5 to 17months were randomized to receive a medicated diet formulated by mixing the RO5461111 cathepsin S inhibitor or a standard diet for at least 12 weeks. Cornea mechanosensitivity was measured with a Cochet-Bonnet esthesiometer. Ocular draining lymph nodes and lacrimal glands (LGs) were excised and prepared for histology or assayed by flow cytometry to quantify infiltrating immune cells. The inflammatory foci (>50 cells) were counted under a 10× microscope lens and quantified using the focus score. Goblet cell density was investigated in periodic acid-Schiff stained sections. Ctss-/- mice were compared to age-matched wild-type mice. Aged mice subjected to cathepsin S inhibition or Ctss-/- mice showed improved conjunctival goblet cell density and cornea mechanosensitivity. There was no change in total LG focus score in the diet or Ctss-/- mice, but there was a lower frequency of CD4+IFN-γ+ cell infiltration in the LGs. Furthermore, aged Ctss-/- LGs had an increase in T central memory, higher numbers of CD19+B220-, and fewer CD19+B220+ cells than wild-type LGs. Our results indicate that therapies aimed at decreasing cathepsin S can ameliorate age-related dry eye disease with a highly beneficial impact on the ocular surface. Further studies are needed to investigate the role of cathepsin S during aging.

Cellulose Acetate Phthalate-Based pH-Responsive Cyclosporine A-Loaded Contact Lens for the Treatment of Dry Eye.

Cyclosporine A (CsA) as an eye drop is an effective treatment for dry eye. However, it has potential side effects and a short ocular residence time. To overcome these obstacles, we developed a cellulose acetate phthalate-based pH-responsive contact lens (CL) loaded with CsA (CsA-CL). The CsA was continuously released from the CsA-CL at physiological conditions (37 °C, pH 7.4) without an initial burst. CsA was well-contained in the selected storage condition (4 °C, pH 5.4) for as long as 90 days. In safety assays, cytotoxicity, ocular irritation, visible light transmittance, and oxygen permeability were in a normal range. CsA concentrations in the conjunctiva, cornea, and lens increased over time until 12 h. When comparing the therapeutic efficacy between the normal control, experimental dry eye (EDE), and treatment groups (CsA eye drop, naïve CL, and CsA-CL groups), the tear volume, TBUT, corneal fluorescein staining at 7 and 14 days, conjunctival goblet cell density, and corneal apoptotic cell counts at 14 days improved in all treatment groups compared to EDE, with a significantly better result in the CsA-CL group compared with other groups (all p < 0.05). The CsA-CL could be an effective, stable, and safe option for inflammatory dry eye.

Involvement of aberrant acinar cell proliferation in scopolamine-induced dry eye mice

Dry eye is a multifactorial disease that causes dryness, inflammation and damage of ocular surface. Subcutaneous injection of the muscarinic cholinergic antagonist scopolamine under desiccating stress reduces tear production and induces dry eye symptoms in mice. However, the expression profile and pathogenic changes of the lacrimal gland remain incompletely understood. In the present study, we performed comparative transcriptomic analysis of lacrimal glands from the control and scopolamine-treated mice. Primary analysis identified 677 upregulated genes and 269 downregulated genes in the lacrimal gland of mice with scopolamine treatment. Unexpectedly, KEGG pathway and hierarchical clustering analysis showed the enrichment of “DNA replication” and “cell cycle” categories in the upregulated genes. Subsequently, we confirmed that the acinar cells were the major proliferating cells of lacrimal gland, which exhibited significant increasing of the proliferating cell nuclear antigen (PCNA) expression after scopolamine treatment, accompanied with the upregulation of DNA damage marker γ-H2AX. More importantly, both prophylactic and therapeutic administration of the cyclin-dependent kinase (CDK) inhibitor AT7519 rescued the tear reduction and alleviated dry eye severity in the scopolamine-treated mice, including corneal epithelial barrier function, lacrimal and corneal inflammation, and conjunctival goblet cell density. Therefore, we conclude that aberrant acinar cell proliferation is involved in the scopolamine-induced tear reduction and dry eye onset, which can be improved by AT7519 treatment.

The In Vivo Effect of a Prostaglandin on the Conjunctival Goblet Cells

Introduction To evaluate the density of conjunctival goblet cells (GCs) from in vivo confocal microscopy (IVCM) and impression cytology (IC) and mucin expression in ten glaucomatous patients treated with preservative-free latanoprost (Monoprost®) Methods Ten glaucomatous patients (10 eyes) were enrolled. Conjunctiva was examined clinically and morphologically by IVCM and IC. Both IVCM and IC were performed at baseline, and after the 1st and 4th month of Monoprost® therapy. GC density (GCD) in cells⁄mm2 was the main outcome measurement. Results At baseline, mean GCD was 147+/-78 cells/mm2 for IVCM and 114+/-62 cells/mm2 for IC GCD. At month one, mean GCD was 149+/-69 and 120+/-56 measured with IVCM and IC, respectively (p=0.31 for IVCM and p=0.40 for IC). At month four, GCD increased to 162+/-81 and 134+/-61 measured with IVCM and IC, respectively (p=0.02 for IVCM and p=0.02 for IC). Conclusion Treatment with preservative-free (PF) latanoprost was associated with an increase in conjunctival GCD in glaucomatous eyes. Further studies are mandatory to verify this finding because its validation may have important consequences in the medical management of glaucoma.

The Therapeutic Effects of a PEDF-Derived Short Peptide on Murine Experimental Dry Eye Involves Suppression of MMP-9 and Inflammation

PurposeTo evaluate the efficacy of a pigment epithelium-derived factor (PEDF)-derived short peptide 29-mer, on the treatment and prevention of experimental dry eye (EDE).MethodsC57BL/6 mice were housed in a low humidity controlled environment chamber for 14 days to induce EDE. The 29-mer was administered topically to their eyes, for treatment or dosing, from the point of housing in the controlled environment chamber. The efficacy of the 29-mer on EDE was evaluated in terms of corneal epithelial integrity, tear secretion, and the density of conjunctival goblet cells. PEDF and inflammatory factors, including tumor necrosis factor-α, IL-1β, IL-6, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase-9, and macrophage infiltration, were examined by real-time polymerase chain reaction, Western blotting, and immunostaining. The involvement of the PEDF receptor/PNPLA2 on the 29-mer effects was evaluated by a specific inhibitor, atglistatin. Rabbit corneal epithelial cells were exposed to hyperosmotic medium to induce inflammatory responses.ResultsThe levels of PEDF protein increased in the corneal epithelium of EDE, compared with the nonstressed mice. The 29-mer showed a therapeutic effect on EDE and prevented the development of EDE, accompanied by amelioration of the inflammatory factors. The 29-mer effects of inflammatory relief were dramatically reversed by atglistatin. The 29-mer also suppressed the expression of matrix metalloproteinase-9 and proinflammatory cytokines in rabbit corneal epithelial cells induced by hyperosmolarity.ConclusionsThrough this animal study, we provide a proof of concept of the anti-inflammatory domain of PEDF having potential to treat dry eye disease.Translational RelevanceThis study shows the 29-mer has novel potential as an ophthalmic drop treatment for dry eye disease.

New, potent, small molecule agonists of tyrosine kinase receptors attenuate dry eye disease.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3) bind to tyrosine kinase (Trk) receptors, TrkA, TrkB, and TrkC, respectively. This study investigated the efficacy of novel molecule agonists of Trk receptors in an in vivo model of dry eye disease (DED). Small molecule TrkC agonist (C1) and a pan-Trk agonist (pan) were synthesized for this. C57BL/6J mice were subjected to desiccating stress (DS) and received bilateral eye drops of C1, pan, or vehicle (2x/day). Dry eye signs, inflammation and expression of corneal barrier function, and conjunctival goblet cell (GC) densities were measured as part of the DED phenotype. Corneal epithelial lysates were collected for either western blot or RNA extraction. Extracted total RNAs were used for NanoString analyses. Immunofluorescent staining was performed on whole-mount corneas using anti-TNFAIP3 and anti-EP4 antibodies. Compared to vehicle, mice subjected to desiccating stress and treated with agonists pan and C1 showed improved corneal barrier function, while C1 also increased GC density. NanoString analyses revealed upregulation of specific mRNA transcripts (Ptger4, Tnfaip3, Il1a and Ptger4, Tlr3, Osal1) in pan- and C1-treated corneas compared to vehicle-treated corneas. Western blots showed that pan and C1 decreased vehicle-induced NFkB nuclear translocation after DS for one day and increased EP4 and TNFAIP3 protein levels after 5 days of DS in corneal epithelium lysates. We conclude that small-molecule agonists of Trk receptors improve DED by decreasing NFkB activation and increasing protein expression of anti-inflammatory molecules TNFAIP3 and EP4. Surprisingly, the most efficacious small molecule agonists were not TrkA selective but TrkC and panTrk, suggesting that wider exploration of TrkB and C and pan Trk agonists are warranted in efforts to treat DED.

Benzalkonium Chloride for Experimental Dry Eye Induction in a Rabbit Model

Purpose: This study compared the clinical parameters and histological findings according to the benzalkonium chloride concentration (BAC; 0.05%, 0.1%, and 0.2%) for inducing experimental dry eye (EDE) in a rabbit model.Methods: Rabbits were divided into four groups according to the BAC concentration: untreated group, 0.05%, 0.1%, and 0.2% BAC. BAC was instilled topically in both eyes of the rabbits twice daily until they were euthanized after 14 days. Tear volume, tear break-up time (TBUT), and corneal fluorescein staining score (CFS) were measured 0, 3, 5, 7, and 14 days after treatment. After excising tissues on day 14, the conjunctival goblet cell density and corneal epithelial apoptosis were quantified.Results: The tear volume and TBUT were lower and the CFS was higher than baseline values after 14, 10, and 5 days in the 0.05, 0.1, and 0.2% BAC groups, respectively (all p &lt; 0.05). At 14 days, the 0.2% BAC group showed more significant aggravation of all clinical parameters, and the 0.1% BAC group had a lower CFS (all p &lt; 0.05) than the 0.05% BAC group. In all BAC groups, the conjunctival goblet cell density was lower and corneal epithelial apoptosis was significantly higher than in the untreated group (all p &lt; 0.01). The conjunctival goblet cell density was lower in the 0.2% BAC group than in the 0.05% BAC group. Between-group differences in corneal epithelial apoptosis were observed in all experimental groups (all p &lt; 0.01).Conclusions: Instilling BAC for 14 days induced EDE in the 0.05, 0.1, and 0.2% BAC groups. Although 0.2% BAC cannot be used for a severe EDE model, it is useful for inducing EDE in a rabbit model in a short time.

The Link module of human TSG-6 (Link_TSG6) promotes wound healing, suppresses inflammation and improves glandular function in mouse models of Dry Eye Disease

PurposeTo investigate the potential of the Link_TSG6 polypeptide comprising the Link module of human TSG-6 (TNF-stimulated gene/protein-6) as a novel treatment for dry eye disease (DED). MethodsWe analyzed the therapeutic effects of topical application of Link_TSG6 in two murine models of DED, the NOD.B10.H2b mouse model and the desiccating stress model. The effects of Link_TSG6 on the ocular surface and DED were compared with those of full-length TSG-6 (FL_TSG6) and of 0.05% cyclosporine (Restasis®). Additionally, the direct effect of Link_TSG6 on wound healing of the corneal epithelium was evaluated in a mouse model of corneal epithelial debridement. ResultsTopical Link_TSG6 administration dose-dependently reduced corneal epithelial defects in DED mice while increasing tear production and conjunctival goblet cell density. At the highest dose, no corneal lesions remained in ∼50% of eyes treated. Also, Link_TSG6 significantly suppressed the levels of inflammatory cytokines at the ocular surface and inhibited the infiltration of T cells in the lacrimal glands and draining lymph nodes. Link_TSG6 was more effective in decreasing corneal epithelial defects than an equimolar concentration of FL_TSG6. Link_TSG6 was significantly more potent than Restasis® at ameliorating clinical signs and reducing inflammation. Link_TSG6 markedly and rapidly facilitated epithelial healing in mice with corneal epithelial debridement wounds. ConclusionLink_TSG6 holds promise as a novel therapeutic agent for DED through its effects on the promotion of corneal epithelial healing and tear secretion, the preservation of conjunctival goblet cells and the suppression of inflammation.

Cathepsin S is a novel target for age-related dry eye

Cathepsin S (Ctss) is a protease that is proinflammatory on epithelial cells. The purpose of this study was to investigate the role of Ctss in age-related dry eye disease. Ctss−/− mice [in a C57BL/6 (B6) background] of different ages were compared to B6 mice. Ctss activity in tears and lacrimal gland (LG) lysates was measured. The corneal barrier function was investigated in naïve mice or after topical administration of Ctss eye drops 5X/day for two days. Eyes were collected, and conjunctival goblet cell density was measured in PAS-stained sections. Immunoreactivity of the tight junction proteins, ZO-1 and occludin, was investigated in primary human cultured corneal epithelial cells (HCEC) without or with Ctss, with or without a Ctss inhibitor. A significant increase in Ctss activity was observed in the tears and LG lysates in aged B6 compared to young mice. This was accompanied by higher Ctss transcripts and protein expression in LG and spleen. Compared to B6, 12 and 24-month-old Ctss−/− mice did not display age-related corneal barrier disruption and goblet cell loss. Treatment of HCEC with Ctss for 48 h disrupted occludin and ZO-1 immunoreactivity compared to control cells. This was prevented by the Ctss inhibitor LY3000328 or Ctss-heat inactivation. Topical reconstitution of Ctss in Ctss−/− mice for two days disrupted corneal barrier function. Aging on the ocular surface is accompanied by increased expression and activity of the protease Ctss. Our results suggest that cathepsin S modulation might be a novel target for age-related dry eye disease.

Comparative analysis of 0.1% cyclosporin A cationic emulsion and 0.05% cyclosporin A emulsion in murine dry eye cases with different severities.

Dry eye (DE), especially severe DE (SDE), can cause ocular surface defects and reduce the patient's quality of life. Several clinical studies have shown that 0.1% cyclosporin A cationic emulsion (CsA CE) could decrease corneal damage. However, no experimental study has reported the effect of 0.1% CsA CE on SDE. The present study aimed to compare the efficacy of 0.1% CsA CE with that of 0.05% CsA emulsion for ocular surface damage and inflammation in the cases of murine DE with different severities. Following exposure to desiccating stress and subcutaneous injection of scopolamine for 5 days, C57BL/6 female mice were divided into SDE and non-SDE (NSDE) groups based on corneal fluorescein staining scores (CFSs). Mice from both groups were topically treated with 0.05% CsA emulsion or 0.1% CsA CE for 10 days. The results demonstrated that 0.1% CsA CE-treated mice in the SDE and NSDE groups exhibited significant improvements in all the clinical and experimental parameters. Furthermore, the CFS of 0.1% CsA CE-treated mice in the SDE group was lower compared with that of the 0.05% CsA-treated mice. In addition, in the SDE group, 0.1% CsA CE-treated mice had significantly lower levels of nuclear factor-κB activation, inflammatory infiltrations and apoptosis on the ocular surface, and they also exhibited higher conjunctival goblet cell density compared with the 0.05% CsA-treated mice. In summary, these findings indicated that 0.1% CsA CE was more effective than topical 0.05% CsA emulsion at improving corneal epithelial injury and decreasing the levels of inflammatory cytokines and T cells in mice with SDE.

Effects of eye drops containing a mixture of 3% diquafosol sodium and tocopherol acetate (vitamin E) on the ocular surface of murine dry eye

Purpose To investigate the efficacy of topical application of 3% diquafosol sodium (DQS) and tocopherol (TCP) acetate mixtures in a mouse model of experimental dry eye (EDE). Methods After exposure to desiccating stress for 5 days, eye drops consisting of 3% DQS alone, 0.01% TCP alone, or 3% DQS and 0.005% or 0.01% TCP mixture were applied for the treatment of EDE. Tear volume, tear film break-up time (TBUT), corneal fluorescein staining scores (CFSS), and tear film lipid layer grades (TFLLG) were measured at 0, 5 and 10 days after treatment. The 2',7'-dichlorodihydrofluorescein diacetate assay (DCFDA) for reactive oxygen species (ROS) production, enzyme-linked immunosorbent assay (ELISA) for malondialdehyde (MDA), and flow cytometry for CD4 + interferon (IFN)-γ+ T cells were evaluated on the ocular surface at 10 days after treatment. In addition, levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and chemokine CC motif ligand 4 (CCL4) in the conjunctiva were measured using a multiplex immunobead assay, and conjunctival goblet cells were counted by periodic acid-Schiff staining at 10 days after treatment. Results Both the TCP mixture groups indicated a significant improvement in TBUT, ROS production, and MDA concentrations compared to those in the DQS alone group. Furthermore, the 0.01% TCP mixture group also showed higher tear film lipid layer grades and conjunctival goblet cell density and lower corneal fluorescein staining scores, number of CD4 + IFN-γ+ T cells, and levels of TNF-α, IL-1β, and CCL4 than the DQS alone group (P < 0.05). Conclusions Application of eye drops containing the mixture of DQS and TCP could stabilize the tear film lipid layer, improve TBUT and corneal epithelial damages, decrease ROS production, inflammatory molecules, and T cells, and increase conjunctival goblet cell density on the ocular surface. Topical DQS and TCP mixtures may have a greater therapeutic effect on clinical signs, oxidative damage, and inflammation of dry eye than DQS eye drops.