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

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

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.

Purpose: to assess the effect of topical administration of the Neurokin-1 receptor (NK1R) antagonist Fosaprepitant in a pre-clinical model of ocular Graft-versus-Host disease (GVHD)., Methods: BALB/c mice were pre-conditioned by myeloablative total body irradiation and subjected to allogeneic bone marrow transplantation and mature T cell infusion (BM + T). BM-transplanted mice (BM) were used as controls. Ocular GVHD was specifically assessed by quantifying corneal epithelial damage, tear secretion, blepharitis and phimosis, 3 times/week for 28 days post-transplantation. A group of BM + T mice received Fosaprepitant 10 mg/mL, 6 times/day, topically, from day 7-29 after transplantation. After sacrifice, the expression of NK1R, CD45, CD3, and CXCL10 was quantified in the cornea, conjunctiva, and lacrimal gland by immunohistochemistry., Results: BM + T mice developed corneal epithelial damage (day 0-29, p < 0.001), blepharitis (day 0-29, p < 0.001), and phimosis (day 0-29, p < 0.01), and experienced decreased tear secretion (day 21, p < 0.01) compared to controls. NK1R was found upregulated in corneal epithelium (p < 0.01) and lacrimal gland (p < 0.01) of BM + T mice. Fosaprepitant administration significantly reduced corneal epithelial damage (p < 0.05), CD45 + (p < 0.05) and CD3 + (p < 0.01) immune cell infiltration in the cornea and conjunctiva (p < 0.001 and p < 0.001, respectively). In addition, Fosaprepitant reduced the expression of CXCL10 in the cornea (p < 0.05) and in the lacrimal gland (p < 0.05)., Conclusions: Our results suggest that NK1R represents a novel druggable pathway for the therapy of ocular GVHD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The pathophysiology of dry eye disease (DED) remains largely unknown, accounting in part for the lack of successful treatments. We explored the pathophysiology of DED using a rabbit model of chronic DED induced with 3 weekly injections of Concanavalin A into the periorbital lacrimal glands. The transcriptome of full-thickness's conjunctival tissue from rabbits with DED and from normal controls was determined using microarrays and, as needed, confirmatory real-time polymerase chain reactions. Results were subjected to bioinformatic analysis. DED induced large-scale changes in gene transcription involving 5,184 genes (22% of the total). Differentially expressed genes could be segregated into: functional modules and clusters; altered pathways; functionally linked genes; and groups of individual genes of known or suspected pathophysiological relevance to DED. A common feature of these subgroups is the breadth and magnitude of the changes that encompass ocular immunology and essentially all aspects of cell biology. Prominent changes concerned innate and adaptive immune responses; ocular surface inflammation; at least 25 significantly altered signaling pathways; a large number of chemokines; cell cycle; and apoptosis. Comparison of our findings to the limited extant transcriptomic data from DED patients associated with either Sjogren's syndrome or non-Sjogren's etiologies revealed a significant correlation between human and rabbit DED transcriptomes. Our data, establishing the large-scale transcriptomic changes of DED and their potential similarity to the human, underscore the enormous complexity of DED; establish a robust animal model of DED; will help expand our understanding of its pathophysiology; and could guide the development of successful therapeutic strategies., Competing Interests: The authors declare no competing interests except for BR who has an equity position in Medicon Pharmaceuticals, Inc. and Apis Therapeutics, LLC; and LH, who has served as an employee of Medicon Pharmaceuticals, Inc. and is currently an employee with an equity position in Apis Therapeutics, LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The glycocalyx is a dense and diverse coat of glycans and glycoconjugates responsible for maintaining cell surface integrity and regulating the interaction of cells with the external environment. Transmembrane mucins such as MUC1 and MUC16 comprise a major component of the epithelial glycocalyx and are currently used to monitor disease progression in cancer. At the ocular surface, multiple lines of evidence indicate that abnormal expression of the enzymes responsible for glycan biosynthesis during pathological conditions impairs the glycosylation of transmembrane mucins. It is now becoming clear that these changes contribute to modify the interaction of mucins with galectin-3, a multimeric lectin crucial for preserving the ocular surface epithelial barrier. This review highlights the potential of using the epithelial glycocalyx as a reliable source for the generation of biomarkers to diagnose and monitor ocular surface disease.

Purpose: To investigate response profiles in the lacrimal functional unit of dry eye disease (DED) and healthy volunteers after exposure to a controlled adverse desiccating environment (CADE) by identifying groups of individuals with similar clinical and molecular changes., Methods: Clinical parameters and tear molecule levels of 20 mild-moderate DED patients and 20 healthy volunteers were evaluated pre- (baseline) and post-CADE exposure. Clustering based on relative change from baseline values was used to identify response profiles. One-vs-all logistic regression was used to identify baseline predictors for response clusters., Results: Four response profiles were identified. Cluster 1: tear break-up time (TBUT) decrease and matrix metalloproteinase 9 (MMP-9) increase. Cluster 2: marked increase in corneal staining, up-regulation of both MMP-9 and interleukin (IL)-6 levels, and down-regulation of epithelial growth factor (EGF). Cluster 3: increase in fractalkine, vascular endothelial growth factor (VEGF), MMP-9, IL-6, IL-8, IL-1 receptor antagonist (IL-1Ra) and RANTES (regulated on activation, normal T expressed and secreted) tear levels; and increased corneal staining and decreased TBUT and phenol red thread scores. Cluster 4: decreased single-item score dry eye questionnaire (SIDEQ) scores and increased corneal staining. Predictive models using baseline variables found that cluster membership depended on: corneal and conjunctival staining, SIDEQ score, interferon gamma-induced protein (IP)-10, VEGF, and IL-1Ra concentrations., Conclusions: The response of both mild-moderate DED and healthy asymptomatic individuals to environmental stress (CADE) can be predicted based on baseline (pre-exposure) clinical and tear molecular parameters. Thus, identifying individuals with a predictable response could improve patient enrollment in DED clinical trials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Dry eye syndrome (DES) is a common symptom of tear film instability and ocular surface damage due to an abnormal quality and quantity of tears, including the sensation of foreign objects and blurred vision. Among all factors for tear film stability, MUC5AC and MUC19 are very important; the levels of both mucins are associated with the pathogenesis of DES., Objectives: The aim of this study was to explore the expression of MUC5AC and MUC19 on the ocular surface in a DES model of ovariectomized female rabbits., Material and Methods: Healthy female New Zealand white rabbits (n = 18; age: 1 year, weight: 2.5 ±0.6 kg) were randomly assigned to a test group and a control group. The DES model was constructed in ovariectomized female rabbits. Indicators of ocular surface injury, such as Schirmer's test, corneal fluorescence staining, a conjunctival imprinting cytology test, and the expression of MUC5AC and MUC19 in conjunctival tissues were evaluated by immunohistochemistry in week 1, week 2 and week 4., Results: Both the length of soaked test paper and the total scores of corneal fluorescence staining at all time-points were significantly lower in the test group than in the control group, and they decreased over time (p < 0.05). The grades of imprinted cells at all time-points were significantly higher in the test group than in the control group, and they increased over time (p < 0.05). The percentage of goblet cells was significantly lower in the test group than in the control group, and it decreased over time (p < 0.05). The percentages of cells with a positive expression of MUC5AC and MUC19 at all time-points were significantly lower in the test group than in the control group, and they decreased over time (p < 0.05)., Conclusions: The pathogenesis of DES is associated with an increased grade of imprinted cells, decreased goblet cells, and a decreased expression of MUC5AC and MUC19.

Woundhealing disorders characterized by impaired or delayed re-epithelialization are a serious medical problem that is painful and difficult to treat. Gelsolin (GSN), a known actin modulator, supports epithelial cell regeneration and apoptosis. The aim of this study was to estimate the potential of recombinant gelsolin (rhu-pGSN) for ocular surface regeneration to establish a novel therapy for delayed or complicated wound healing. We analyzed the influence of gelsolin on cell proliferation and wound healing in vitro, in vivo/ex vivo and by gene knockdown. Gelsolin is expressed in all tested tissues of the ocular system as shown by molecular analysis. The concentration of GSN is significantly increased in tear fluid samples of patients with dry eye disease. rhu-pGSN induces cell proliferation and faster wound healing in vitro as well as in vivo/ex vivo. TGF-β dependent transcription of SMA is significantly decreased after GSN gene knockdown. Gelsolin is an inherent protein of the ocular system and is secreted into the tear fluid. Our results show a positive effect on corneal cell proliferation and wound healing. Furthermore, GSN regulates the synthesis of SMA in myofibroblasts, which establishes GSN as a key protein of TGF-β dependent cell differentiation.

Purpose: The S100 fused-type proteins hornerin (HRNR) and filaggrin-2 (FLG2) are members of the epidermal differentiation complex, which is involved in terminal differentiation of keratinocytes via cornification as well as maintenance of the epidermal antimicrobial barrier. We investigated the expression and possible regulation of HRNR and FLG2 at the ocular surface and in the lacrimal apparatus., Methods: Tissues of the lacrimal apparatus and ocular surface were analyzed systematically by means of RT-PCR, immunohistochemistry, and immuntransmission electron microscopy (iTEM) for their ability to express and produce HRNR and FLG2. In addition, inducibility and regulation of HRNR were studied in cultivated human corneal (HCE), conjunctival (HCjE), as well as meibomian gland (HMGEC) epithelial cell line by real-time RT-PCR., Results: RT-PCR, immunohistochemistry, and iTEM revealed constitutive expression of HRNR in the epithelium of cornea, conjunctiva, nasolacrimal ducts, and acinus cells of lacrimal and meibomian glands. HRNR also was detected in tears of healthy volunteers. No expression of FLG2 could be detected in tissue samples of the ocular surface and lacrimal apparatus. Real-time RT-PCR revealed a decreased HRNR gene expression after challenge with proinflammatory cytokines and supernatants of Escherichia coli and Pseudomonas aeroginosa in HCE cells, whereas HCjE cells revealed no changes. In HMGECs serum-induced differentiation and application of all-trans retinoic acid significantly increased HRNR gene expression., Conclusions: The data suggest that HRNR, but not FLG2, is a component of the ocular surface and lacrimal apparatus, including meibomian glands. HRNR seems to contribute to the maintenance of the epithelial barrier at the ocular surface and, thus, also may be involved in ocular surface diseases.

Dry eye is a complicated ocular surface disease whose exact pathogenesis is not yet fully understood. For the therapeutic evaluation and pathogenesis study of dry eye, we established an in vitro three-dimensional (3D) coculture model for the ocular surface. It is composed of rabbit conjunctival epithelium and lacrimal gland cell spheroids, and recapitulates the aqueous and mucin layers of the tear film. We first investigated the culture conditions for both cell types to optimize their secretory functions, by employing goblet cell enrichment, air-lifting culture, and 3D spheroid formation techniques. The coculture of the two cell components leads to elevated secretion and higher expression of tear secretory markers. We also compared several coculture systems, and found that direct cell contact between the two cell types significantly increased tear secretion. Inflammation was induced to mimic dry eye disease in the coculture model system. Our results showed that the coculture system provides a more physiologically relevant therapeutic response compared to monocultures. Our work provides a complex 3D model as a recapitulation of the ocular surface and tear film system, which can be further developed as a model for dry eye disease and therapeutic evaluation.

Purpose: The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics., Materials and Methods: ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers., Results: Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES., Conclusions: Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

The ocular surface system is responsible for ensuring that the precorneal tear film is sufficient in both quality and quantity to preserve optimal vision. Tear secretion is a complex, multifactorial process, and dysfunction of any component of the ocular surface system can result in tear film instability and hyperosmolarity with resultant dry eye disease. The tear film is primarily composed of lipids, aqueous, and mucins, with aqueous accounting for most of its thickness. The aqueous is produced by the main lacrimal gland, accessory lacrimal glands, and corneal and conjunctival epithelia. Although the main lacrimal gland has long been considered an indispensable source of the aqueous component of tears, there is evidence that adequate tear secretion can exist in the absence of the main lacrimal gland. We review and discuss the basics of tear secretion, the tear secretory capacity of the ocular surface, and emerging treatments for dry eye disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: This study reports tear compensation observed in rabbits with bilateral resection of main lacrimal gland (LG) and explored the potential mechanisms., Methods: Dry eye conditions were created by resection of nictitating membrane (NM), Harderian gland (HG), and main LG in eight (16 eyes) male New Zealand White rabbits. In addition to Schirmer test, Periodic acid-Schiff (PAS) staining, and ocular surface staining with fluorescein and rose Bengal, conjunctival impression cytology was employed before and up to 4 months after excision (AE). Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), expression of inflammatory biomarkers (IL-1β, TNF-α, and matrix metalloproteinase-9) were monitored. Further, involvement of ionic and water transporters were investigated in conjunctival epithelium., Results: Significant dry eye phenotypes in rabbits were observed 1 month AE, which corroborated with elevated biomarker mRNA expression. However, Schirmer test score and goblet cell numbers never decreased AE in conjunctival epithelium. Moreover, ocular surface staining, and biomarker expression declined to baseline in over 4 months AE. No upregulation was observed of the following conjunctival ionic transporters: cystic fibrosis transmembrane conductance regulator, sodium potassium chloride cotransporters, sodium potassium ATPase, and epithelial sodium channels. Instead, aquaporin (AQP) 4 and AQP5 were upregulated. Immunolocalization and immune blotting of AQP4 was demonstrated in rabbit conjunctival epithelium., Conclusions: In the absence of NM, HG, and main LG in rabbits, tear secretion was not decreased and significant improvement of dry eye phenotypes observed with time AE. Conjunctival AQPs are possibly involved in a compensatory tear fluid production.

Purpose: P2Y2 receptors are expressed on ocular surface tissues. Diquafosol ophthalmic solution (DIQUAS(®) ophthalmic solution 3 %; Santen Pharmaceutical Co., Ltd.) acts on these receptors and promotes the secretion of water and mucin. It has been shown to be an efficient dry eye treatment. If P2Y2 receptor expression on the ocular surface decreases with age, the effect of diquafosol may be reduced in elderly persons. In this study, we investigated the changes in P2Y2 receptor expression on the rat ocular surface over an extended period of time., Methods: P2Y2 receptor expression in the conjunctiva, cornea, meibomian gland and lacrimal glands of male and female Sprague-Dawley rats was examined from 5 weeks until 53 weeks of age using immunostaining and quantitative-PCR., Results: In the immunohistological examinations, P2Y2 receptor expression was observed in the conjunctival epithelium containing goblet cells, corneal epithelium, meibomian gland ductal epithelium and lacrimal gland ductal epithelium. However, its expression was not significantly different between each age group or between sexes. Regarding P2Y2 receptor mRNA expression, there was an age-related increase in the bulbar conjunctiva. In particular, a significant increase was observed in the 53-week-old age group as compared to the 5-week-old female age group. However, age-related changes in expression were not observed in the cornea or meibomian gland in males or females., Conclusions: We observed no significant age-related decrease was observed for P2Y2 receptor protein and mRNA expression on rat ocular surface tissues.

Importance: Lubricin may be an important barrier to the development of corneal and conjunctival epitheliopathies that may occur in dry eye disease and contact lens wear., Objective: To test the hypotheses that lubricin (ie, proteoglycan 4 [PRG4 ]), a boundary lubricant, is produced by ocular surface epithelia and acts to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink and that lubricin deficiency increases shear stress on the ocular surface and promotes corneal damage., Design, Setting, and Participants: Human, porcine, and mouse tissues and cells were processed for molecular biological, immunohistochemical, and tribological studies, and wild-type and PRG4 knockout mice were evaluated for corneal damage., Results: Our findings demonstrate that lubricin is transcribed and translated by corneal and conjunctival epithelial cells. Lubricin messenger RNA is also present in lacrimal and meibomian glands, as well as in a number of other tissues. Absence of lubricin in PRG4 knockout mice is associated with a significant increase in corneal fluorescein staining. Our studies also show that lubricin functions as an effective friction-lowering boundary lubricant at the human cornea-eyelid interface. This effect is specific and cannot be duplicated by the use of hyaluronate or bovine serum albumin solutions., Conclusions and Relevance: Our results show that lubricin is transcribed, translated, and expressed by ocular surface epithelia. Moreover, our findings demonstrate that lubricin presence significantly reduces friction between the cornea and conjunctiva and that lubricin deficiency may play a role in promoting corneal damage.

Dry eye is a common ocular surface disease of multifactorial etiology characterized by elevated tear osmolality and inflammation leading to a disrupted ocular surface. The latter is a risk factor for ocular surface infection, yet overt infection is not commonly seen clinically in the typical dry eye patient. This suggests that important innate mechanisms operate to protect the dry eye from invading pathogens. This article reviews the current literature on epidemiology of ocular surface infection in dry eye patients and laboratory-based studies on innate immune mechanisms operating at the ocular surface and their alterations in human dry eye and animal models. The review highlights current understanding of innate immunity in dry eye and identifies gaps in our knowledge to help direct future studies to further unravel the complexities of dry eye disease and its sequelae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OS(Wt)), expression of dnMaml1 at the ocular surface (OS(dnMaml1)) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OS(dnMaml1) phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OS(dnMaml1) dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.

The aim of the study was to elucidate aquaporin (AQP) family member mRNA expression and protein expression/localization in the rat lacrimal functional unit. The mRNA expression of all rat AQPs (AQP0-9, 11-12) in palpebral, fornical, and bulbar conjunctiva, cornea, lacrimal gland, and Meibomian gland was measured by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and real time RT-PCR. Antibodies against AQP1, 3, 4, 5, 9, and 11 were used in Western blotting and immunohistochemistry to determine protein expression and distribution. Our study demonstrated characteristic AQP expression profiles in rat ocular tissues. AQP1, 3, 4, 5, 8, 9, 11, and 12 mRNA were detected in conjunctiva. AQP0, 1, 2, 3, 4, 5, 6, 11, and 12 mRNA were expressed in cornea. AQP0, 1, 2, 3, 4, 5, 7, 8, and 11 mRNA were detected in lacrimal gland. AQP1, 3, 4, 5, 7, 8, 9, 11, and 12 mRNA were identified in Meibomian gland. By Western blot, AQP1, 3, 5, and 11 were detected in conjunctiva; AQP1, 3, 5, and 11 were identified in cornea; AQP1, 3, 4, 5, and 11 were detected in lacrimal gland; and AQP1, 3, 4, 5, 9, and 11 were present in Meibomian gland. Immunohistochemistry localized AQPs to distinct sites in the various tissues. This study rigorously analyzed AQPs expression and localization in rat conjunctiva, cornea, lacrimal gland, and Meibomian gland tissues. Our findings provide a comprehensive platform for further investigation into the physiological or pathophysiological relevance of AQPs in ocular surface., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the effect of the topical steroid, fluorometholone, and the non-steroidal anti-inflammatory drugs (NSAIDs), nepafenac and ketorolac, on inflammatory cytokine expression of the ocular surface in the botulium toxin B-induced murine dry eye model., Methods: Topical artificial tears (0.5% carboxymethylcellulose sodium), 0.1% fluorometholone, 0.1% nepafenac, and 0.4% ketorolac were applied 3 times per day in a dry eye mouse model 1 week after intralacrimal botulium toxin B (BTX-B) or saline (sham) injection. Tear production and corneal fluorescein staining were evaluated in all groups before injection at baseline and at 3 time points up to 4 weeks after injection. The pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evaluated by immunohistochemistry., Results: BTX-B-injected mice showed significantly decreased aqueous tear production and increased corneal fluorescein staining at the 1 and 2 week time points compared with normal control and saline-injected mice. In the BTX-B-injected mice, immunofluorescent staining for TNF-α and IL-1β in corneal and conjunctival epithelial cells increased significantly at the 2 and 4 week time points compared to that of normal and saline-injected mice, and returned to normal levels at the 4 week time point. Topical fluorometholone significantly improved corneal surface staining in the BTX-B-injected mice after 1 week of treatment, and increased the tear production within 2 weeks, but without statistical significant difference. Topical fluorometholone significantly decreased the staining of TNF-α and IL-1β in corneal and conjunctival epithelia after 1-week treatment. Topical artificial tears, 0.1% nepafenac, and 0.4% ketorolac did not show obvious effects on tear production, corneal surface staining, and levels of IL-1β and TNF-α expression in normal, and BTX-B-injected dry eye mice., Conclusions: Topical fluorometholone caused suppression of inflammatory cytokine expression on the ocular surface in the Botulium toxin B-induced murine dry eye model, while topical NSAIDs demonstrated no clearly beneficial effects.

Purpose: Psoriasin, originally isolated from psoriasis as an overexpressed molecule of unknown function, has recently been identified as a principal Escherichia coli-killing antimicrobial peptide of healthy skin. The purpose of this study was to investigate the expression and antimicrobial role of psoriasin at the ocular surface and in the lacrimal apparatus., Methods: Different tissues of the lacrimal apparatus and ocular surface were systematically analyzed by means of RT-PCR, Western blot, and immunohistochemistry for their ability to express and produce psoriasin. The inducibility and regulation of psoriasin were studied in human corneal as well as conjunctival epithelial cell lines after challenge with ocular pathogens and proinflammatory cytokines. Real-time RT-PCR was performed to evaluate the expression and induction of psoriasin. In addition, tear fluid obtained from different healthy volunteers was examined by ELISA for its psoriasin concentration., Results: RT-PCR and Western blot analyses revealed a constitutive expression of psoriasin in cornea, conjunctiva, nasolacrimal ducts, and lacrimal gland. Immunohistochemistry showed strong staining of meibomian glands for psoriasin. No induction of psoriasin was observed after stimulation with supernatants of E. coli, whereas supernatants of Staphylococcus aureus and Haemophilus influenzae significantly increased the psoriasin mRNA expression. Stimulation with IL-1β and VEGF also strongly increased psoriasin transcription. The highest amounts of psoriasin protein were detected in the tear fluid (~170 ng/mL) of healthy volunteers., Conclusions: The results suggest that psoriasin is produced by the structures of the ocular surface and is part of the innate immune system at the ocular surface and tear film.

Purpose: Epidemiological studies suggest that dietary n-3 polyunsaturated fatty acids (PUFAs) may protect against dry eye. This study aimed to evaluate whether a dietary deficiency in n-3 PUFAs may increase the severity of the pathology in a scopolamine-induced model of dry eye in the rat., Methods: Lewis rats of three consecutive generations were bred under a balanced diet or a diet deprived of n-3 PUFAs. Dry eye was experimentally induced by continuous scopolamine delivery in female animals from the third generation of both groups. After 10 days of treatment, the clinical signs of ocular dryness were evaluated in vivo using fluorescein staining. MHC II and the rat mucin rMuc5AC were immunostained on ocular sphere cryosections. The transcript levels of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were quantified in the exorbital lacrimal glands (LG) and in the conjunctiva using reverse transcription followed by polymerase chain reaction. Lipids were extracted from the exorbital LG for fatty acid analysis of the phospholipids using gas chromatography., Results: When compared to control animals, the scopolamine treatment induced an increase in the cornea fluorescein staining score (from 0.5 ± 0.0 to 2.5 ± 1.0 arbitrary units (AU) for the balanced diet and from 1.2 ± 0.8 to 2.6 ± 0.5 AU for the n-3 PUFA-deficient diet); a decrease in rMuc5AC immunostaining in the conjunctival epithelium (-34% for the balanced diet and -23% for the n-3 PUFA-deficient diet); an increase in the LG transcript levels of TNF-α for the balanced diet and of TNF-α and IFN-γ for the deficient diet; an increase in the conjunctival transcript levels of IL-1β and IL-6 for the deficient diet; an increase in arachidonic acid (AA) and in the ∆5-desaturase index (ratio of AA to dihomo-gamma-linolenic acid) in the exorbital LG for both diets. When compared to the balanced diet, the n-3 PUFA-deficient diet induced an increase in the LG transcript levels of IL-6 for the control animals and of TNF-α for the control and dry eye animals as well as an increase in the conjunctival transcript levels of IL-6 for the dry eye animals. There was no significant diet difference in fluorescein staining, rMuc5AC, and MHC II immunostaining scores., Conclusions: Our data suggest that an n-3 PUFA deficiency does not increase the severity of dry eye in a rat model of dry eye.

Background: To present experience with conjunctivocystorhinostomies (CDCR) at pediatric age., Methods: Operative and postoperative data were collected for 15 pediatric CDCRs (aged 9 to 14.5 years) performed endonasally. Four procedures were performed bilaterally, seven unilaterally. The follow-up examinations were made every 3 months in the first year, every 6 months in the second year, and every 12 months afterwards. The patient s satisfaction with the surgery, lacrimal drainage function, and complications were evaluated. The drainage function of the lacrimal system was assessed on the basis of the history, clinical examinations, and the fluorescein dye disappearance test. The data were taken from the medical records and written questionnaires., Results: After the surgery, 12 children were very satisfied, two children partially satisfied, and one child was not satisfied (due to the loss of the tube). Full or partial functional success was found in 14 cases (93.3%); no functional success was observed in one case. The number of complications was higher than in other studies concerning adult patients. A total of 37 complications were observed in our study; most of the complications occurred in more than one of the children, and were observed in some children several times. The most frequent complications were lateral or medial malposition (25 times) and the extrusion of the tube (eight times). It was necessary to re-insert the tube in seven cases under general anaesthesia. The complications mostly occurred within the first 3 months after the surgery (seven complications during the first week, 12 complications between the 2nd and the 4th week, eight complications between the 5th and the 12th week, and three complications between the 9th and the 12th postoperative week)., Conclusions: CDCR appears to be a reasonable procedure in children over 10 years old. Experience in lacrimal surgery (pediatric dacryocystorhinostomy, CDCR in adults), and good cooperation and compliance of children are a fundamental assumption for successful pediatric CDCR.

This study investigated the expression of MUC19, a newly discovered gel-forming mucin gene, in normal human lacrimal functional unit components and its alteration in Sjögren syndrome patients. Real-time PCR and immunohistochemistry were performed to determine the expression of MUC19 and MUC5AC in human cornea, conjunctiva, and lacrimal gland tissues. Conjunctival impression cytology specimens were collected from normal control subjects and Sjögren syndrome patients for Real-time PCR, PAS staining, and immunohistochemistry assays. In addition, conjunctiva biopsy specimens from both groups were examined for the expression differences of MUC19 and MUC5AC at both mRNA and protein level. The MUC19 mRNA was found to be present in cornea, conjunctiva and lacrimal gland tissues. The immunohistochemical staining of mucins showed that MUC19 was expressed in epithelial cells from corneal, conjunctival, and lacrimal gland tissues. In contrast, MUC5AC mRNA was only present in conjunctiva and lacrimal gland tissues, but not in cornea. Immunostaining demonstrates the co-staining of MUC19 and MUC5AC in conjunctival goblet cells. Consistent with the significant decrease of mucous secretion, both MUC19 and MUC5AC were decreased in conjunctiva of Sjögren syndrome patients compared to normal subjects. Considering the contribution of gel-forming mucins to the homeostasis of the ocular surface, the decreased expression of MUC19 and MUC5AC in Sjögren syndrome patients suggested that these mucins may be involved in the disruption of the ocular surface homeostasis in this disease.

Purpose: To evaluate the expression and presence of the surfactant proteins (SP) B and C in the lacrimal apparatus at the ocular surface and in tear fluid., Methods: Expression of SP-B and SP-C was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, meibomian gland, accessory lacrimal glands, cornea, and nasolacrimal ducts. The deposition of the hydrophobic proteins SP-B and SP-C was determined by Western blot and immunohistochemistry in healthy tissues, tear fluid, and aqueous humor., Results: The presence of both SP-B and SP-C on mRNA and protein level was evidenced in healthy human lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts. Moreover, both proteins were present in tear fluid but were absent in aqueous humor. Immunohistochemical investigations revealed production of both peptides by acinar epithelial cells of the lacrimal gland and additionally by accessory lacrimal glands of the eyelid as well as epithelial cells of the conjunctiva and nasolacrimal ducts. Immunohistochemically, healthy cornea and goblet cells revealed no reactivity., Conclusions: Besides the recently detected surfactant-associated proteins SP-A and SP-D, our results show that SP-B and SP-C are also peptides of the tear film, the ocular surface, and the lacrimal apparatus. Based on the current knowledge of lowering surface tension in alveolar lung cells, a similar effect of SP-B and SP-C may be assumed concerning the tear film.

Purpose: Hormone diseases induce changes in the lacrimal gland (LG) and ocular surface (OS). Thyroid hormone (TH) induces cell proliferation and lipid metabolism through the activation of TH receptors. The aim of the present study was to evaluate the location and comparative expression of TH receptor beta-1 (Thrb) in LG of rats with hypothyroidism and in controls and to evaluate the impact of this disease on LG and OS structure and function., Methods: Hypothyroidism was induced in Wistar male rats by the long-term use of tiamazole. Ten weeks later corneal cells were collected for impression cytology (IC). Rats were humanely killed, and tissues were evaluated by immunoperoxidase staining and Western blot for Thrb. The content of malondialdehyde (MDA) and acetylcholine (ACh) in LG was determined by spectrophotometry (n = 5/group in all experiments)., Results: LG weight was significantly lower in hypothyroid rats (P < 0.05). Western blot analysis indicated that LGs express Thrb and that hypothyroidism induces a higher expression of this receptor. IC was significantly different and ACh was significantly lower in hypothyroid rats (P < 0.05)., Conclusions: Chronically reduced levels of TH lead to biochemical and structural changes and modulate the levels of Thrb in LG. These events confirm that LG is a target organ for TH and may facilitate understanding of the mechanism related to dry eye in hypothyroidism.

Purpose: Mucins are highly glycosylated proteins that act as lubricants, protectants, and mediators of signal transduction. The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family members initiate mucin-type O-glycosylation. Because O-glycosylation provides mucins with the viscoelastic properties required for proper mucin function, ppGaNTase expression is vital to the maintenance of healthy epithelial surfaces including the ocular surface. Differences of expression of ppGaNTase isoforms might be factors influencing susceptibility to dry eye disease. Therefore, we determined the expression of the ppGaNTase isoforms in normal human ocular surface tissues and the conjunctival epithelium from patients with aqueous-deficient dry eye., Methods: Expression of ppGaNTase isoforms was quantitated by real-time reverse transcriptase polymerase chain rection (RT-PCR)., Results: Conjunctiva and cornea expressed multiple ppGaNTase isoforms, with isoforms T12 and T4 being the most strongly expressed in conjunctiva and T12 and T3 in the cornea. In contrast, lacrimal gland expressed fewer isoforms and had lower total ppGaNTase expression. Brush cytology was found to be superior to impression cytology for harvesting conjunctival epithelium in terms of ease of use, safety, and reproducibility of results. Similar to whole conjunctiva, the strongest isoforms in conjunctival epithelial cells were T12 and T4, followed by T3, T1, T5, and T2. No significant differences of ppGaNTase expression were found between the conjunctival epithelium of dry eye and normal control groups., Conclusion: Human ocular surface tissues express multiple ppGaNTase isoforms, suggesting a requirement for glycosylating diverse mucin-type substrates. However, there is no evidence to date to suggest that differences of ppGaNTase expression levels might contribute to susceptibility to dry eye disease.

This study was performed to evaluate the changes of tear film and ocular surface caused by smoking. Symptom scoring, tear film break-up time (BUT), basal tear secretion test, corneal sensitivity test, keratoepitheliopathy scoring, and conjunctival impression cytology were performed in 29 smokers (58 eyes) and 26 non-smokers (52 eyes). Tear film BUT, basal tear secretion, corneal sensitivity, and squamous metaplasia were 7.71+/-2.66 sec, 6.29+/-2.85 mm, 53.69+/-5.69 mm, and 2.45+/-1.26 in smokers and 9.62+/-3.14 sec, 10.04+/-3.87 mm, 56.46+/-4.79 mm, and 1.12+/-0.83 in non-smokers, respectively (p<0.05). Symptom score, keratoepitheliopathy score, and goblet cell density were not significantly different between the two groups. Tear film BUT was shorter, basal tear secretion and corneal sensitivity were lower, and squamous metaplasia was higher in heavy smokers than in light smokers. In conclusion, smoking deteriorates the tear film and ocular surface with decreased quantity and quality of tear film, decreased corneal sensitivity, and squamous metaplasia, and this deterioration is related to the amount of smoking.

Mucins are vital for maintenance of a healthy, wet ocular surface. Once only thought to be secreted by goblet cells, mucins are now also known to be of the membrane-associated type. Stratified ocular surface epithelia express at their apical-tear fluid surface a repertoire of membrane-associated mucins including MUC1, MUC4, MUC16. These mucins are concentrated on the tips of the microplicae, forming a dense glycocalyx at the epithelial tear film interface. A major mucin of the secretory class is the goblet-cell-derived gel-forming mucin MUC5AC. A small soluble mucin, MUC7, is expressed by the lacrimal gland acini. Our hypothesis of the role/distribution of the secreted and membrane-associated mucins at the ocular surface is that the secreted mucins are soluble in the tear fluid, and are moved about and shunted to the nasolacrimal duct and by the eyelids during blinking. Thus, in the tears, the secreted mucins act as clean-up/debris removing multimeric networks that at the same time, through their hydrophilic nature, hold fluids in place and harbor defense molecules secreted by the lacrimal gland. Membrane-associated mucins, on the other hand, form a dense barrier in the glycocalyx at the epithelial tear film interface. This barrier prevents pathogen penetrance and is a lubricating surface that allows lid epithelia to glide over the corneal epithelia without adherence. The secreted mucins move easily over the glycocalyx mucins because both have anionic character that creates repulsive forces between them. Little is known regarding regulation of expression and glycosylation of mucins by ocular surface epithelia. Since ocular surface drying diseases alter both goblet cell and mucin production, and production and glycosylation of membrane-associated mucins, studies of mucin gene regulation and glycosylation may yield treatment modalities for these diseases.

Introduction: Components of the mucosal immune system (MALT) have been identified in the conjunctiva (as CALT) and the lacrimal drainage system (as LDALT). Their structural and functional relation with the established immune protection by the lacrimal gland is unclear., Material and Methods: Macroscopically normal and complete tissues of the conjunctiva, lacrimal drainage system and lacrimal gland from human body donors were investigated by analysis of translucent whole mounts, and using histology, immunohistology as well as scanning and transmission electron microscopy., Results: A typical diffuse lymphoid tissue, composed of effector cells of the immune system (T-lymphocytes and IgA producing plasma cells) under an epithelium that contains the IgA transporter SC, is not isolated in the conjunctiva and lacrimal drainage system. It is anatomically continuous from the lacrimal gland along its excretory ducts into the conjunctiva and from there via the lacrimal canaliculi into the lacrimal drainage system. Lymphoid follicles occur in a majority (about 60%) and with bilateral symmetry. The topography of CALT corresponds to the position of the cornea in the closed eye., Conclusion: These results show that the MALT of the lacrimal gland, conjunctiva and lacrimal drainage system constitute an anatomical and functional unit for immune protection of the ocular surface. Therefore it should be integrated as an "eye-associated lymphoid tissue" (EALT) into the MALT system of the body. EALT can detect ocular surface antigens by the lymphoid follicles and can supply other organs and the ocular surface including the lacrimal gland with specific effector cells via the regulated recirculation of lymphoid cells.

Purpose: Several mucins including MUC1, MUC2, MUC4, and MUC5AC have been identified at the ocular surface and in tears. The lacrimal gland, however, is not generally considered a source of ocular mucin. Because the lacrimal glands are similar to the salivary glands, we hypothesized that the lacrimal gland would express MUC7, a distinctive salivary mucin. We report the presence of MUC7 RNA and protein in normal human lacrimal glands as determined by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Western blot analysis., Methods: RNA from lacrimal glands and conjunctivae was isolated and subjected to RT-PCR with primers specific for MUC7. The identity of the PCR products was confirmed by sequencing. In situ hybridization with PCR product-based riboprobes was used to locate MUC7 transcripts in the lacrimal gland. MUC7 protein was detected by Western blot analysis., Results: Of six normal human lacrimal glands from which relatively intact mRNA could be extracted, four expressed MUC7. Hybridization with an antisense riboprobe for MUC7 indicates the presence of MUC7 transcripts in the cytoplasm of acinar cells. Western blot analysis confirms expression of the protein in the lacrimal gland. The presence of MUC1, MUC4, and MUC5B was also demonstrated by RT-PCR in lacrimal gland tissue. MUC7 transcripts and protein were also detected in normal human conjunctivae., Conclusions: The mucin profile of the lacrimal gland resembles that of the salivary gland. Both RNA and protein corresponding to MUC7 are present in the normal human lacrimal gland. Reverse transcription-polymerase chain reaction indicates that other transcripts of MUC1, MUC4, and MUC5B are present as well. Ocular MUC7 is also produced by the conjunctival mucosa. The lacrimal gland, therefore, contributes not only to the aqueous component of tears but also, in concert with the conjunctiva, may contribute to the total pool of ocular surface mucins.

Purpose: To review the significance of mucin in the tear film and the ocular surface epithelium., Methods: Summary of the information on how mucin derived from the corneal and conjunctival epithelia and from goblet cells plays a role in the stability of the tear film over the ocular surface. The change in mucin expression derived from the ocular surface epithelium is also discussed with reference to ocular surface disease., Results: The corneal and conjunctival epithelia produce transmembrane mucins such as MUC1, MUC2, and MUC4. In contrast, goblet cells produce the gel-forming secretory mucin, MUC5AC. The lacrimal gland produces MUC7. On the ocular surface, cooperation between transmembrane mucin and secretory mucin is necessary for the stability of the tear film. The expression of mucin from the ocular surface epithelium is coordinated from the time of eyelid opening and is altered in conditions such as squamous metaplasia and dry eye. This alteration may result in instability of the tear film. CONCLU SION: The induction of mucin from the ocular surface may facilitate the stability of the tear film, and increased knowledge may lead to the development of a new modality for the treatment of dry eye.

Objective: To evaluate the effect of partial surgical excision of enlarged lacrimal caruncles, termed megalocaruncles, in patients with epiphora from functional lacrimal drainage obstruction., Design: Retrospective, noncomparative, interventional case series., Participants: From May 1995 through December 1999, 88 patients (46 women and 42 men; mean age, 68 years) with epiphora, patent lacrimal drainage system on irrigation, and a megalocaruncle underwent a partial lacrimal carunculectomy procedure. In 26 patients, a bilateral partial lacrimal carunculectomy was performed., Intervention: Lacrimal caruncular tissue, with its overlying mucosa, was partially excised, and the wound was closed with interrupted sutures., Main Outcome Measures: Symptomatology and patient satisfaction., Results: After a partial lacrimal carunculectomy procedure, epiphora was subjectively improved in 77% of the patients, ranging from complete relief in 33% to significant improvement in 44% of the patients. Twenty-three percent of the patients had no change in symptoms. The mean follow-up was 12.7 months., Conclusions: Megalocaruncles can be the cause of epiphora in patients with functional lacrimal drainage obstruction, for whom a partial lacrimal carunculectomy is an effective surgical procedure in 77% of the cases.

Purpose: To evaluate the effect of temporary punctal occlusion on tear production, tear clearance, and ocular surface sensation in normal subjects., Methods: Noncomparative interventional case series. Punctal occlusion with silicone punctal plugs was performed on nine normal subjects without complaints of ocular irritation and no known history of ocular surface disease. The lower punctum of both eyes was occluded in five subjects. The upper and lower puncta of only one eye were occluded in four subjects. Corneal and conjunctival sensations were measured with the Cochet-Bonnet anesthesiometer. Tear fluorescein clearance was evaluated with a CytoFluor II fluorophotometer by measuring the fluorescein concentration in minimally stimulated tear samples collected from the inferior tear meniscus 15 minutes after instillation of fluorescein. Schirmer test was performed without anesthesia. The tests were performed at days 0, 1, 3, 7, and 14 to 17 after punctal occlusion. Relationships were analyzed with linear regressions, and a quadratic term was used to model a return to preocclusion levels. Paired t test was used to study the change in tear fluorescein concentration., Results: In subjects who had the lower puncta of both eyes occluded, conjunctival sensation decreased in both eyes (right eye, P =.008; left eye, P =.003), but there was no change in corneal sensation. Their tear fluorescein clearance did not show a significant change from baseline (P =.90). However, a decrease in Schirmer test scores approached statistical significance (P =.056). In subjects with both puncta of only one eye occluded, we noted a decrease in corneal sensation (occluded eye, P =.042; nonoccluded eye, P =.036), conjunctival sensation (occluded, P =.001; nonoccluded, P =.060), and Schirmer scores (occluded, P =.022; nonoccluded, P =.011). Linear regression did not show a significant change in tear fluorescein clearance for either eye (occluded, P =.28; nonoccluded, P =.44). However, paired t test showed a significant worsening of tear clearance in the occluded eye from day 0 to day 3 (P =.001) followed by a subsequent improvement in tear clearance from day 3 to the end of the study period (P =.045). Paired t test did not reveal any significant changes in tear clearance in the nonoccluded eye. The quadratic term of the linear regression model demonstrated an increase toward preocclusion levels that approached statistical significance for corneal sensation (occluded, P =.053; nonoccluded, P =.099). It was statistically significant for conjunctival sensation (occluded, P =.001; nonoccluded, P =.045) and Schirmer scores (occluded, P =.047; nonoccluded, P =.044)., Conclusions: Temporary punctal occlusion in normal subjects decreases tear production and ocular surface sensation. Our findings suggest that in addition to blocking tear drainage, punctal occlusion may affect the ocular surface/lacrimal gland interaction. These effects were more pronounced in subjects with both upper and lower puncta occluded. In normal subjects, there appears to be an autoregulatory mechanism to return tear production, tear clearance, and ocular surface sensation to preocclusion levels 14 to 17 days after punctal occlusion.

Background/aims: The antimicrobial activity of the tear film exceeds the activity of its known constituents. The authors postulate that this excess activity is the result of antimicrobial peptides called defensins, and they aimed to look for defensins in the human eye., Methods: Evidence of defensin production was sought by reverse transcriptase polymerase chain reaction (RT-PCR). Intron spanning primers were designed for beta defensins 1 and 2, and alpha defensins 5 and 6. RT-PCR was performed on cornea, conjunctiva, and lacrimal gland samples, and reaction products were size fractionated and sequenced to confirm their identity. A monoclonal antibody was utilised for the detection of alpha defensins 1, 2, and 3 in tissue sections and in immunoblots of tears., Results: RT-PCR revealed beta defensin 1 message in samples of conjunctiva, cornea, and lacrimal gland. beta Defensin 2 message was detected in the conjunctiva and cornea but was absent from the lacrimal gland. alpha Defensin 5 and 6 message was absent in these tissues but alpha defensins 1, 2, and 3 were detected in normal tears, lacrimal gland, and inflamed conjunctiva by immunochemistry., Conclusion: The data suggest the human eye innately produces a spectrum of antimicrobial defensin peptides. Defensins hold therapeutic potential in ocular infections as they have a broad spectrum of antimicrobial activity (bacteria fungi and viruses ) and accelerate epithelial healing.

Purpose and Background: To measure azithromycin levels in rabbit lacrimal and Harder glands, conjunctiva and plasma after a single oral dose of 20 mg/kg. Drug levels in lacrimal gland tissue are significant in trachoma because the gland may be involved in the disease process and it is the source of tears by which the drug is carried to the external eye., Methods: Lacrimal and Harder glands, conjunctiva and plasma were collected from New Zealand white female rabbits at 24, 48, 72, 96 and 144 h following a single oral dose of azithromycin (20 mg/kg). Azithromycin levels in tissue and plasma were measured using high-performance liquid chromatography (HPLC) electrochemical detection., Results: Azithromycin levels peaked within the first 24 h in all tissues and plasma assayed. The highest concentration was in the lacrimal gland (6.2 microg/g, SD +/- 0.8), followed by Harder gland (4.4 microg/g, SD +/- 0.8), conjunctiva (0. 9 microg/g, SD +/- 0.5) and plasma (0.06 microg/g, SD +/- 0.03). These concentrations reached their lowest measured levels at 120 and 144 h., Conclusion: Azithromycin levels measured throughout the 144 h after dosing were consistently above the minimum inhibitory range (MIC) for Chlamydia trachomatis (0.03-0.25 microg/ml) in the lacrimal glands, while the conjunctiva maintained a concentration above the MIC for 96 h and stayed within MIC levels for 144 h.

Purpose: The exact etiology of dry eye is unknown but is believed to be multifactorial. Apoptosis has been implicated in the pathogenesis of autoimmune diseases such as Sjögren's syndrome (SS). This study attempted to gain a better understanding of the role of apoptosis and its regulation in the patho-physiology of dry eye. The therapeutic effect of immunomodulatory agents such as cyclosporin A (CsA) in the treatment of dry eye, particularly its impact on the level of apoptosis in the target tissues, is also investigated., Methods: A colony of dogs with spontaneous chronic idiopathic keratoconjunctivitis sicca (KCS) was maintained. Nictitans lacrimal gland (NLG), an accessory lacrimal gland, and conjunctival biopsies of the KCS and normal dogs were obtained before and after 12 weeks of treatment with 0.2% topical CsA ophthalmic emulsion b.i.d. (Allergan, Inc., Irvine, CA, U.S.A.). Tissues were prepared for the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) apoptosis assay and immunohistochemical analysis for various apoptosis mediators., Results: The TUNEL assay demonstrated that (i) the normal NLG and conjunctival epithelial cells exhibited a limited level of apoptosis; (ii) in KCS dogs, lacrimal acinar and conjunctival epithelial cells underwent an increased apoptosis, whereas the lymphocytes had a significantly lower level of apoptosis compared to those of the normal dogs; (iii) after topical CsA, apoptosis was induced in the lymphocytes and suppressed in the acinar and conjunctival epithelial cells in KCS dogs. Immunohistochemistry revealed that p53, fas, and fasL, but not bcl-2 were highly expressed in the target tissues of KCS dogs. The immunoreactivity of p53 was significantly decreased, whereas the bcl-2 level was increased after CsA administration., Conclusions: The induction of epithelial cell apoptosis and the suppression of lymphocytic apoptosis in the NLG and ocular-surface tissues, such as conjunctiva of KCS dogs, indicates the important role of this phenomenon in the etiology of dry eye. Topical CsA appears to facilitate lymphocytic apoptosis and suppress epithelial cell apoptosis in the KCS dog. The differential expression of various apoptotic mediators after topical treatment implicates CsA in facilitating the reestablishment of the normal apoptotic balance, suggesting additional mechanisms by which CsA is therapeutic for dry-eye syndrome.

Four different types of lectin were applied to the rabbit conjunctiva and the lacrimal sac to examine the distribution of glycoconjugates. The conjunctival epithelium is comprised of goblet cells and nongoblet cells. The mucus granules of the goblet cells were stained with soybean agglutinin (SBA), and the cell body of the nongoblet cells was labeled with concanavalin A (Con A). The glycocalyx of the apical surfaces of the goblet cells and of the non-goblet cells was labeled with Maculura pomifera agglutinin. The lacrimal sac mucosa is comprised of superficial light and dark cells, and basal cells. The cell bodies of the light cells were stained with SBA. The glycocalyx of the apical surfaces of the light and dark cells was characteristically labeled with Con A. These results suggest that the composition of glycoconjugates is markedly different between the conjunctiva and the lacrimal sac, especially in the cell surface glycocalyx.

Purpose: This work describes the snake-like chromatin cells in the conjunctiva of patients with keratoconjunctivitis sicca (KCS), assessing the relationship between tear function parameters and the severity of the disease, and searching for any differential pattern that could lead to early and diagnosis help clarify the etiology of KCS., Methods: The conjunctiva of 366 eyes from 187 patients with KCS, and 30 eyes from 15 controls, were classified according to the results of the Schirmer I test, break-up time, rose Bengal staining, osmolarity, ferning tests and electrophoresis; they were then studied by impression cytology and electron microscopy., Results: Snake-like chromatin cells, other nuclear changes, and a decreased number of goblet cells were found by impression cytology in different areas of the KCS conjunctiva. The presence of snake-like chromatin was significantly correlated with the severity of the disease. No nuclear changes were found in the control group., Conclusions: We assume these findings can be attributed to a natural regeneration of conjunctival cells and to resistance to a pathological reaction to KCS.

Background: Increasing evidence indicates that long-term use of topically administered medication can induce changes in the conjunctiva and lachrymal function., Methods: In order to evaluate changes in the conjunctiva and lachrymal response after prolonged use of topically administered antiglaucoma medications and preservatives found in antiglaucomatous medication solutions (benzalkonium chloride), we tested lachrymal function (Schirmer I., Jones, BUT, Ferning tests) and used the conjunctival impression cytology technique., Materials: A group of patients with primary open angle glaucoma (POAG) receiving topical antiglaucomatous medication were recruited. A second group received only preservative instillations while a control group was formed of similarly aged subjects with no eye disease and was given topical or systemic medical therapy. Excluded from the trial were patients with a history of external eye disease or who had received conjunctival surgery., Results: Tear secretion was reduced against that of the control group in those subjects who received protracted administration of antiglaucomatous eyedrops (timolol and/or pilocarpine). A statistically significant degree of conjunctival metaplasia was associated with long-term use of topical medication. The subjective symptoms reported by those patients receiving chronic topical antiglaucomatous therapy and the objective observations on them were found to be proportional to the observed tearing response. Changes were more pronounced in subjects who received only benzalkonium chloride., Conclusion: Our study results suggest that long-term use of antiglaucoma medication induces changes in both tear film and conjunctival surface. Such changes may be related to the medication or the duration of treatment, but may also be due to the preservatives used in the commercial product.

The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial cell populations in normal human ocular mucosa-associated lymphoid tissue (MALT; LG, conjunctiva, and cornea) based on their immunohistochemical staining patterns with anticytoskeletal monoclonal antibodies (MoAbs) reactive with cytokeratins (AE-1, AE-2, AE-3, AE-5, AE-14, PKK1, and 34 beta E12), muscle-specific actin (HHF35), and vimentin. AE-1 stained LG (acini, ducts, and myoepithelia) and the full thickness of corneal and conjunctival epithelia. It stained only the superficial and basal limbal epithelium. AE-2 weakly stained all epithelia, except LG acini and proximal intralobular ducts. AE-3 and 34 beta E12 MoAbs had strong immunoreactivity with all MALT epithelia. AE-5 strongly stained the inner cells (suprabasal) of LG central intra- and interlobular ducts and the suprabasal epithelial layers of the cornea. It weakly stained LG myoepithelia and the superficial conjunctival epithelium. AE-14 stained the outer (basal) cells of LG central intra- and interlobular ducts, LG myoepithelia, basal epithelial layers of the limbus and conjunctiva, and all corneal epithelia. PKK1 stained all epithelia, except the basal limbus. HHF35 and the antivimentin MoAbs stained only the LG myoepithelia. The results of these studies indicate that the different epithelia in human ocular MALT may be differentiated by specific patterns of immunoreactivity with anticytoskeletal MoAbs. These MoAbs may be useful molecular markers for identifying ocular MALT epithelia.

Autoradiographic studies were performed to determine the localization of irreversibly bound radioactivity in the eyes and accessory structures of mice exposed to 14C-labelled organic solvents in vivo or in vitro. A selective localization of bound radioactivity was observed in the conjunctival epithelium of mice given i.v. injections of 1,2-dibromoethane, chloroform, carbon tetrachloride or bromobenzene. Similar results were observed after instillation of chloroform or carbon tetrachloride in the conjunctival sac and after incubation of eyelids with the labelled compounds in vitro. A high level of irreversibly bound radioactivity was also observed in the excretory ducts of the intraorbital lacrimal glands of mice exposed to 1,2-dibromoethane in vivo and in vitro. After incubation of 14C-labelled 1,2-dibromoethane or chloroform with homogenates prepared from rat conjunctiva, the presence of irreversibly protein-bound radioactivity was detected. The results indicate that the conjunctival epithelium can metabolically activate halogenated organic solvents into products that bind to the tissue. The significance of a metabolic activation of chemicals in the pathogenesis of chemically induced lesions in the conjunctiva merits further attention.

Literature on human plasma concentrations after instillation of ocular timolol, levobunolol, atropine, cyclopentolate, scopolamine, phenylephrine, betamethasone and technetium Tc 99m and theories of lacrimal drainage were reviewed. In all studies the eyedrops absorbed rapidly into the systemic circulation. Like the kinetics of the tracer substances in lacrimal scintigraphy, the plasma drug levels showed interindividual variations. Plasma levels of ocular drugs were lower when punctal occlusion was applied, the mechanism, however, could not be explained. Since an early and a late plasma peak was occasionally registered in some subjects in timolol and cyclopentolate studies, it is suggested that systemic absorption of ocular drugs is low during the nasolacrimal passage but occurs during conjunctival and nasal contact.

Elimination of technetium (pertechnetate in normal saline solution) was studied from various types of contact lenses placed on normal human eyes by means of computer assisted gamma camera using "region of interest technique" with the designated area corresponding to the conjunctival sac. An elimination 4 times slower was found from a highly hydrophilic lens (Scanlens) than from a HEMA-lens (Softlens), 0,5% min and 2.0% min, respectively. From an ultrathin lens (U3-Softlens) was eliminated 2.4% min. Hard lenses did not absorb the isotope. In a laboratory study the lenses were pre-soaked in pertechnetate, blotted and washed at 2 min intervals in 0.5 ml saline. By this procedure 3% min of the technetium was eliminated from Scanlens, 16% min from Softlens and 28%/min from a thin, therapeutic lens (Plano-T). The ratio Softlens/Scanlens was in the human study 4.0 and in the laboratory study 4.9. Radioactivity was very rapidly eliminated from CAB-lenses. A similar study was carried out with radioactive water. More than 100% min was eliminated in the first 10 min followed by a slower elimination from 10-20 min. Then an increased elimination was seen for a few min. This increased elimination was in repeated studies constantly found after 20 min. It was not found in the studies with technetium and labelled leucine.