Comparison of the Effectiveness between Sampling Methods for Protein Analysis of Tear Fluids

Purpose : Although various sampling methods of tears from conjunctival sac have been reported, no previous study compared their effectiveness or efficiency based on protein extraction. By comparing the compliance, volume and protein concentration of each tear sampling method, we searched for the most efficient tear collection method. Methods : Resting tear samples of 14 eyes of normal subjects were collected using Schirmer paper, capillary tube, cellolose acetate rod and 3 different ophthalmic sponges made of different materials and density (Merocel®, KeraCel® and Weck-Cel®). After centrifugation of the collected tear samples, the tear volume and protein concentration were measured for each method. Additionally, the compliance of each tear sampling method was analyzed by numerically representing the amount of discomfort experienced during resting tear collection. Results : The average volume retrieved by each tear sampling method was 9.0 ± 1.1 µL with no significant differences between groups. The average concentration of protein retrieved by each tear sampling method was 5.3 ± 1.2 µg/µL. Merocel® retrieved 7.6 ± 0.61 µg/µL, which was significantly higher than other sampling methods (p < 0.05). The compliance of Merocel® and the capillary tube were the highest, while KeraCel® showed the lowest compliance. Conclusions : Merocel® retrieved the highest amount of protein and showed high compliance and may be the most effective and easily applicable tear sampling method in clinical settings.ope

Activation of HIF-1alpha (hypoxia inducible factor-1a) prevents dry eye-induced acinar cell death in the lacrimal gland

The pathogenesis of immune-mediated lacrimal gland (LG) dysfunction in Sjögren’s syndrome has been thoroughly studied. However, the majority of dry eye (DE) is not related to Sjögren type, and its pathophysiology remains unclear. The purpose of this study was to determine and investigate the protective mechanisms against DE stress in mice. DE induced prominent blood vessel loss without apoptosis or necrosis in the LG. Autophagic vacuoles, distressed mitochondria, and stressed endoplasmic reticulum were observed via electron microscopy. Immunoblotting confirmed the increase in autophagic markers. Glycolytic activities were enhanced with increasing levels of succinate and malate that, in turn, activated hypoxia-inducible factor (HIF)-1α. Interestingly, the areas of stable HIF-1α expression overlapped with COX-2 and MMP-9 upregulation in LGs of DE-induced mice. We generated HIF-1α conditional knockout (CKO) mice in which HIF-1α expression was lost in the LG. Surprisingly, normal LG polarities and morphologies were completely lost with DE induction, and tremendous acinar cell apoptosis was observed. Similar to Sjögren’s syndrome, CD3+ and CD11b+ cells infiltrated HIF-1α CKO LGs. Our results show that DE induced the expression of HIF-1α that activated autophagy signals to prevent further acinar cell damage and to maintain normal LG function.ope

Comparison of Ocular Surface Mucin Expression After Topical Ophthalmic Drug Administration in Dry Eye-Induced Mouse Model

PURPOSE: To determine the mucinogenic effect of dry eye (DE) treatment drugs currently in use, we compared the levels of mucin production and inflammatory cytokine expression on the ocular surfaces using a DE-induced mice model. METHODS: C57BL/6 mice were separated into 6 groups: a control group, DE-induced mice with the vehicle and treated with cyclosporine A (CsA), rebamipide (Reb), diquafosol tetrasodium (DQS), or prednisolone (Pred). The mRNA expression of MUC 1, 4, 16, 5AC, and proinflammatory cytokines on the corneal epithelia were determined by quantitative real-time polymerase chain reaction. Expression of each MUC was evaluated using flow cytometry and immunohistostaining. Conjunctival goblet cells were analyzed through periodic acid-Schiff (PAS) staining. RESULTS: Desiccating stress significantly decreased both mRNA and protein levels of all MUCs in the cornea. CsA mainly enhanced MUC5AC, with an increase in PAS-positive cells, whereas DQS chiefly increased membrane-associated mucins (MM). However, Reb only minimally increased expression of MUC5AC and Pred only increased MUC4. MUC16 did not show any significant change in any group. On the contrary, the mRNA levels of interleukin (IL)-1β, -6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were increased in the DE corneas of the control mice and were reduced by all treatments; in particular, IL-6 was significantly suppressed. CONCLUSION: Topical DQS and CsA not only ameliorated ocular surface inflammation under desiccating stress but also upregulated both MM and secretory mucins (SM) and contributed to conjunctival goblet cell recovery, compared to Reb and Pred. Both anti-inflammatory and secretory factors should be considered simultaneously when measuring the treatment effect of DE drugs.restrictio

Corneal lymphangiogenesis facilitates ocular surface inflammation and cell trafficking in dry eye disease

PURPOSE: While the normal cornea has limited innervation by the lymphatic system, chronic immune-inflammatory disorders such as dry eye (DE) can induce lymphangiogenesis in the ocular surface. Using a conditional knock-down murine model, Lyve-1Cre;VEGFR2flox mice, this study investigated the role of lymphangiogenesis in the pathophysiology of DE. METHODS: DE was induced in both wild type (WT) B6 and Lyve-1Cre;VEGFR2flox mice. Tissue immunostaining and volumetric gross measurements were used to assess changes in the ocular surface, skin, and lymph nodes (LNs). The expression of lymphangiogenic factors (TNF-α, IL-6/-8/-12/-17, VEGF-C/-D, IFN-γ, VEGFR-2/-3, Lyve-1, and podoplanin) and the frequency of immune cells (CD4, CD11b, and CD207) on the ocular surface and lacrimal glands were quantified by real-time polymerase chain reaction and flow cytometry. RESULTS: Compared to WT mice, there were fewer lymphatic vessels and a reduction in lymphangiogenic markers in the ocular surface and skin of Lyve-1Cre;VEGFR2flox mice. After DE induction, mRNA levels of TNF-α, IL-8, and IFN-γ were significantly reduced in Lyve-1Cre;VEGFR2flox mice compared to WT mice (p < .01). Surprisingly, the LNs from Lyve-1Cre;VEGFR2flox mice with DE were significantly smaller and populated by fewer dendritic cells and effector T cells than those from WT mice (p < .001). Furthermore, immunostaining showed corneal nerves in the DE-induced Lyve-1Cre;VEGFR2flox mice were notably intact like in the naïve condition. CONCLUSIONS: Inhibition of lymphangiogenesis in the cornea effectively attenuates not only the inflammatory response including trafficking of immune cells but also preserves corneal nerves under desiccating stress. Corneal lymphangiogenesis might be a contributing factor in deterioration on the ocular surface homeostasis.restrictio

Dry eye-induced CCR7+CD11b+ cell lymph node homing is induced by COX-2 activities

PURPOSE: We aimed to determine the role of CCR7+CD11b+ cell lymph node (LN) homing and T-cell differentiation in dry eye (DE)-induced immunopathogenesis and investigate the therapeutic effects of cyclooxygenase-2 (COX-2) and prostaglandin E2/eicosanoid-prostanoid (PGE2/EP) inhibitors against DE. METHODS: Six-week-old female C57BL/6 mice were housed in a controlled-environment chamber and administered topical selective COX-2 inhibitors or EP2 antagonists. Expression of major histocompatibility complex (MHC)-IIhigh, CD11b+, CCR7+, IFN-γ+, IL-17+, and CD4+ in the corneas and draining LNs was evaluated using flow cytometry. Mixed lymphocyte reactions (MLRs) with carboxyfluorescein diacetate succinimidyl ester labeling and intracellular cytokine staining were used to verify DE-induced corneal dendritic cell function. mRNA expression of COX-2, EPs, and proinflammatory cytokines in ocular surface was evaluated using quantitative RT-PCR and immunohistochemical staining. RESULTS: Dry eye significantly increased MHC-IIhighCD11b+ and CCR7+CD11b+ cells in the cornea and LNs, and MLR revealed CCR7+CD11b+ cells from DE corneas stimulated IL-17+CD4+ cell proliferation. mRNA levels of COX-2, EP2, IFN-γ, TNF-α, IL-6, and IL-17 were significantly higher in DE ocular surface but were suppressed by topical COX-2 inhibitors and EP2-specific blockers. Immunohistochemical staining showed COX-2 and matrix metalloproteinase expression in DE corneal epithelia that was diminished by both topical treatments. Furthermore, both topical treatments significantly reduced frequencies of MHC-IIhigh, CD11b+, and CCR7+CD11b+ cells in the corneas and LNs, but also IL-17+CD4+ cells in LNs. CONCLUSIONS: Topical COX-2/EP2 treatment reduces CCR7+CD11b+ cells on the ocular surface with inhibition of cellular LN homing and suppresses Th17 immune response, suggesting the COX-2/PGE2/EP axis contributes to immuno-inflammatory pathogenesis on the ocular surface and may be a novel therapeutic target in DE.ope