Search

Purpose: We illustrate the importance of small quantities (<10 mol%) of polar phospholipids on the surface-active behavior of meibum-like lipid compositions., Methods: Artificial meibum-like lipid mixture containing cholesteryl and wax esters was mixed with differing amounts of phosphatidylcholine (PC). The surface activity of these mixtures was investigated at the air-water interface by recording surface pressure created by the lipid layer as a function of molecular area at 37°C. The PC proportion in the mixtures was 0, 2.5, 5, 7.5, or 10 mol%, and the remaining proportion in the mixture was 50:50 (mol/mol) of cholesteryl oleate (CO) and behenyl oleate (BO). Also, the effect of temperature was investigated., Results: The surface activity of the mixtures increased in a very predictable and consistent fashion as a function of the PC proportion. The lipid mixture containing only CO and BO showed miniscule surface activity. However, already 2.5% PC mixed with the nonpolar CO and BO generated considerable increase in surface pressure. At small surface areas, the behavior of 7.5% and 10% PC compositions started to approach that of a pure PC monolayer. The temperature did not have a considerable impact on the surface-active behavior of the PC-containing compositions., Conclusions: The polar phospholipids have a considerable effect on the surface-active properties of artificial tear film lipid layer (TFLL) compositions. Surprisingly, this takes place already at very low and physiologically relevant PC proportions. The effect is more dependent on the actual amount of the phospholipids at the air-tear interface than on the relative amount of these lipids in TFLL.

Purpose: Tear film lipid layer (TFLL) is constantly exposed to reactive ozone in the surrounding air, which may have detrimental effects on ocular health. Behenyl oleate (BO), a representative tear film wax ester, was used to study the reaction with ozone at the air-water interface., Methods: Time-dependent changes in mean molecular area of BO monolayers were measured at different ozone concentrations and surface pressures. In addition, the effect of ascorbic acid on the reaction rate was determined. Reaction was followed using thin-layer chromatography and reaction products were identified using liquid chromatography-electrospray ionization mass spectrometry (LC-MS). Tear fluid samples from healthy subjects were analyzed with LC-MS for any ozonolysis reaction products., Results: Behenyl oleate was found to undergo rapid ozonolysis at the air-water interface at normal indoor ozone concentrations. The reaction was observed as an initial expansion followed by a contraction of the film area. Ascorbic acid was found to decrease the rate of ozonolysis. Main reaction products were identified as behenyl 9-oxononanoate and behenyl 8-(5-octyl-1,2,4-trioxolan-3-yl)octanoate. Similar ozonolysis products were not detected in the tear fluid samples., Conclusions: At the air-water interface, unsaturated wax esters react readily with ozone in ambient air. However, no signs of ozonolysis products were found in the tear fluid. This is most likely due to the antioxidant systems present in tear fluid. Last, the results show that ozonolysis needs to be controlled in future surface chemistry studies on tear film lipids.

Purpose: The tear film lipid layer is believed to stabilize the tear film and to retard evaporation. Based on previous simple in vitro studies, the evidence for the latter property is scarce. In this study, we used complex lipid mixtures including various wax esters to study their physical properties and evaporation retarding effect., Methods: Twelve samples of artificial tear film lipid layer mixtures composed of (L-α)-phosphatidylcholine, cholesterol oleate, and triglycerides were mixed with wax esters. A Langmuir balance was used to analyze the compressibility and rheological properties of these mixtures. In addition, a custom-built system was used for the evaporation studies used at 35°C. Lipid films were imaged with Brewster angle microscopy., Results: None of the studied lipid mixtures decreased the evaporation rate. All lipid mixtures had similar compression isotherms and viscoelastic properties regardless of the wax ester species or its concentration. The results suggest that the overall properties of these mixtures are independent of individual lipid species and that these films are very cooperative and showed minor variation depending on the wax ester species. Brewster angle microscopy images revealed that the lipid films assembled into multiple layers., Conclusions: Wax ester-containing lipid mixtures resembling the tear film lipid layer are organized in a layered fashion so that amphiphilic lipids are adjacent to the aqueous phase and the nonpolar lipids are layered on top of these. This organization does not retard evaporation and raises overall questions about the role of lipids in the tear film., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Since homeostasis at the ocular surface requires a delicate balance between numerous factors, and the external environment contributes as an unpredictable component, we aimed to understand the role that various lipids and their regulators have in the complex process that maintains a healthy corneal surface., Methods: Through basic proteomics, we tested the presence of sphingolipid metabolism enzymes in normal human tears, and then used a cell culture model to study how the proteins are secreted and for what purpose., Results: When studying healthy tears, we found that sphingolipid-specific enzymes, acid and neutral sphingomyelinases, and ceramidases can be detected. The role played by sphingolipid metabolism in stress provided the motivation for further studies concerning their secretion/leakage in the extracellular environment in a cell culture model of human corneal epithelial cells (HCE). Among the stress agents investigated (i.e., ultraviolet B [UV-B] radiation, hyperosmolarity [HO], and lipopolysaccharide [LPS]), UV-B and HO induced dose-dependent release/secretion of sphingomyelinases from the cells. In an attempt to identify the route of secretion or release of the enzyme, we discovered that the tested stress stimuli induced shedding of extracellular vesicles in the HCE-conditioned medium., Conclusions: Extracellular stress affects tear fluid composition more profoundly than just secretion of proinflammatory mediators. Lipids at the ocular surface, either in tear fluid or within the corneal epithelial cells, can be modified by a relatively large array of lipases to modulate their functions. Moreover, extracellular vesicles in the tear fluid could represent a valuable noninvasive diagnosis tool for anterior segment diseases.

Purpose: We examined in vitro the evaporation-retarding effect of wax esters (WEs). The WEs resembled closely the most abundant WE species in meibum., Methods: A custom-built system was used to measure the evaporation rates through WE layers applied to the air-water interface at 35°C and, as a reference, at 30°C and 41°C. Additionally, the melting points of the WEs were determined. The organization and stability of the WE layers were assessed using Brewster angle microscopy (BAM) and Langmuir film experiments, respectively., Results: Four of 19 WEs retarded evaporation at 35°C: behenyl palmitoleate (BP), behenyl oleate (BO), behenyl linoleate (BLN), and behenyl linolenate (BLNN) decreased evaporation by 20% to 40%. BP was the most effective evaporation retardant. At 30°C the most effective retardants were BLN and BLNN decreasing evaporation by ~50%, whereas BP and BO decreased evaporation by only 5% to 10%. At 41°C, each lipid decreased evaporation by only 2% to 4%. The evaporation-retardant WEs all melted within 2°C of physiological temperature. BAM images showed that the evaporation-retardant WE layers spread somewhat uniformly and possibly exhibited areas of condensed lipid. The isotherms suggested that WE layers were surface pressure tolerant but unstable under compression-relaxation cycles., Conclusions: The evaporation-retarding effect is dependent on the physicochemical properties of the WEs at given temperature, and therefore, the effect most likely arises from a certain phase of the WE layer. However, WEs as such are poor surfactants and need to be accompanied by polar lipids to form stable lipid layers.

Purpose: We examined in vitro the potential evaporation-retarding effect of the tear film lipid layer (TFLL). The artificial TFLL compositions used here were based on the present knowledge of TFLL composition., Methods: A custom-built system was developed to measure evaporation rates at 35°C. Lipids were applied to an air-water interface, and the evaporation rate through the lipid layer was defined as water loss from the interface. A thick layer of olive oil and a monolayer of long-chain alcohol were used as controls. The artificial TFLLs were composed of 1 to 4 lipid species: polar phosphatidylcholine (PC), nonpolar cholesteryl ester, triglycerides, and wax ester (WE). Brewster angle microscopy (BAM) and interfacial shear rheometry (ISR) were used to assess the lateral structure and shear stress response of the lipid layers, respectively., Results: Olive oil and long-chain alcohol decreased evaporation by 54% and 45%, respectively. The PC monolayer and the four-component mixtures did not retard evaporation. WE was the most important evaporation-retardant TFLL lipid (∼20% decrease). In PC/WE mixtures, an ∼90% proportion of WE was required for evaporation retardation. Based on BAM and ISR, WE resulted in more condensed layers than the non-retardant layers., Conclusions: Highly condensed, solid-like lipid layers, such as those containing high proportions of WEs, are evaporation-retardant. In multi-component lipid layers, the evaporation-retardant interactions between carbon chains decrease and, therefore, these lipid layers do not retard evaporation.

Purpose: Climatic droplet keratopathy (CDK) is a degenerative disease of the cornea with possible involvement from matrix metalloproteinases (MMPs). Therefore, the authors investigated histologic distribution, levels, and molecular forms of MMP-2 and MMP-9, as well as tear fluid levels of MMPs and cytokines in CDK patients. They additionally examined UV-B-irradiation effect on production of gelatinases and cytokines by human corneal epithelial (HCE) cell culture model., Methods: Tears were collected from 20 unrelated individuals (10 with CDK and 10 controls). CDK affected corneas were haematoxylin-eosin stained and the presence and distribution of MMP-2 and MMP-9 was examined using immunohistochemistry. Gelatinases and cytokine secretion was measured in tears and supernatants from UV-B-exposed HCEs by immunoblotting, gelatin zymography, and protein array, respectively., Results: MMP-2 and MMP-9 values were significantly higher in tears collected from CDK patients than healthy controls and were accompanied by pro-inflammatory cytokine secretion. Immunohistochemistry showed that MMP-2 was expressed at the basement membrane zone in both control and affected corneas, but also marked the edges of the granular CDK deposits; MMP-9 expression was restrained to basal layers of the epithelium and was markedly induced in CDK corneas. In HCE cells, UV-B increased gelatinase secretion, with a striking effect on MMP-9, and was preceded by pro-inflammatory cytokine release., Conclusions: The authors demonstrate that the corneal epithelium could participate in CDK development as a source of cytokines and gelatinases. Additionally, in HCE cells, UV-B- modulated cytokine and subsequent MMP secretion. Local inhibition of cytokine secretion and gelatinases may prevent CDK progression.

Purpose: To determine the changes in corneal sensitivity to different stimulus modalities in diabetes mellitus (DM)1 and DM2 patients with retinopathy, and to explore whether argon laser photocoagulation exacerbates sensitivity loss in diabetic patients., Methods: Corneal sensitivity to different modalities of stimulus was determined in one randomized eye in 52 patients with DM1 (n = 35) or DM2 (n = 17), and in 27 healthy subjects. Medical history was obtained from all the patients, including age, sex, time from DM diagnosis, type of diabetes, time from onset of retinopathy, type of diabetic retinopathy, and type of argon laser treatment. Corneal sensitivity was determined using a gas esthesiometer. Mechanical, chemical, and thermal (heat and cold) stimuli were applied on the central cornea., Results: Sensitivity thresholds to selective mechanical, chemical, and cold stimulation were significantly higher in DM patients compared to controls. Sensitivity threshold to mechanical and chemical stimuli was higher in DM2 than in DM1 patients. In DM1 patients, mechanical threshold increased with time after DM diagnosis. No correlation was found between sensitivity thresholds to chemical or thermal stimulation and the age of the patient, type of retinopathy, or time from its diagnosis. Laser treatment generated a further impairment of corneal sensitivity., Conclusions: Corneal sensitivity to mechanical, chemical, and thermal stimulation is decreased in DM patients, suggesting that diabetes affects homogeneously the different types of sensory neurons innervating the cornea. Corneal sensitivity appears to be more disturbed in DM2 than in DM1. Laser treatment of DM patients generates a further impairment in corneal sensitivity, probably as the result of physical damage to ciliary nerves.

Purpose: To determine corneal sensitivity to selective mechanical, chemical, and thermal (heat and cold) stimulation in patients with a history of herpes simplex virus (HSV) keratitis., Methods: Corneal sensitivity to different modalities of stimulus was determined in both eyes of 16 patients with unilateral HSV keratitis diagnosed 1 to 12 months before the study. On slit lamp examination, 13 HSV-affected eyes showed corneal scarring or opacities, and three had no signs of previous keratitis. Corneal sensitivity was determined with the Belmonte gas esthesiometer. Mechanical, chemical, heat, and cold stimuli were applied on the central cornea. Eyes from 10 healthy subjects served as controls., Results: In all control and contralateral eyes, selective mechanical, chemical, heat, and cold stimulation evoked sensations of subjective intensity proportional to the magnitude of the applied stimulus. In one HSV patient, the affected cornea was unresponsive to all types of stimuli, four lost only corneal sensitivity to mechanical stimulation, and three lost only sensitivity to heat. Mechanical (P<0.005) and heat (P<0.05) thresholds were raised in HSV eyes, whereas thresholds for CO2 were not modified. Also, HSV subjects identified poorly the intensity of mechanical, chemical, and heat stimuli, whereas sensitivity to cold stimulation was unaffected., Conclusions: In eyes that had had HSV keratitis, corneal sensitivity to mechanical forces and heat was significantly impaired, suggesting that axonal damage and/or altered expression of membrane ion channels involved in transduction and membrane excitability affects primarily the mechano- and polymodal nociceptor terminals. Corneal cold-sensitive terminals remain largely unaffected.

Purpose: The principal lipid-interacting protein in human tears has been reported to be tear lipocalin (Tlc). Tlc has been suggested to scavenge harmful lipophilic substances from the corneal epithelium and to maintain the integrity of the anterior tear film lipid layer by binding and releasing lipid(s) that are accommodated within the protein. Although lipids can be extracted from Tlc, it is still unclear whether Tlc can actually bind to lipid membranes and accept membrane lipids and whether it possesses lipid transfer activity. The purpose of this study was to explore the interaction of Tlc with neutral, anionic, and cationic lipid membranes and to assess the potential of Tlc to facilitate the transfer of either polar or neutral lipids in a lipid transfer assay., Methods: The binding of Tlc to lipid membranes was assessed by a monolayer technique and fluorescence spectroscopy. The polar lipid transfer activity of Tlc was assessed with a radiometric assay based on the transfer of (14)C-phosphatidylcholine (PC) from PC-liposomes to HDL(3). The neutral lipid transfer activity of Tlc was assayed by measuring the transfer of radioactive cholesteryl ester from LDL to HDL(3)., Results: Purified Tlc showed significant surface activity as evidenced by an increase in surface pressure at the air-buffer interface. Likewise, it interacted actively with neutral, anionic, and cationic lipid monolayers, as evidenced by an equal increase in surface pressure despite the surface charge. Enhanced quenching of the single tryptophan residue of Tlc by pyrene and I(-) anion suggested that different protein domains are involved in the interaction of Tlc with oppositely charged lipid membranes. Finally, radiometric assays revealed that Tlc does not possess any neutral or polar lipid transfer activity between lipid vesicles or/and lipoproteins., Conclusions: Tlc interacted with lipid membranes composed of neutral, cationic, or anionic membranes, which supports a role for Tlc in the maintenance of the tear film interfaces. Tlc did not show any neutral or polar lipid transfer activity whatsoever. The findings suggest that the notion of the role of Tlc as the major lipid-transferring protein in human tears should be revised.

Purpose: Matrix metalloproteinases (MMPs) play a central role in the wound-healing process. The objective of this study was to identify and characterize the levels and molecular forms of human tear fluid collagenase-2 (MMP-8) and membrane type 1-MMP (MT1-MMP or MMP-14) in patients who had undergone excimer laser photorefractive keratectomy (PRK) and in healthy subjects. Whether MT1-MMP activates pro-MMP-8 was also determined., Methods: Tear fluid samples were collected with scaled and blunted microcapillaries from healthy control subjects and, on the second postoperative day, from patients who had undergone PRK. Time and the volume collected were registered. Molecular forms and levels of pro and active MMP-8 and MT1-MMP in these samples were determined by Western immunoblot analysis, quantitated by computer scanning. The concentration of MMP-8 was also determined by immunofluorescence assay. The conversion of pure human polymorphonuclear neutrophil (PMN) pro-MMP-8 to the active form by the catalytic domain of MT1-MMP was studied by Western immunoblot analysis., Results: The tear fluid flow was increased after PRK. Tear fluid flow-corrected excretion of MMP-8 was significantly higher in PRK-treated patients, as assessed by immunofluorescence assay and quantitative Western immunoblot analysis. The major MMP-8 species detected in tears of both PRK-treated patients and healthy control subjects represented latent and active 75- and 65-kDa highly glycosylated MMP-8 isoforms. The less-glycosylated 45- to 55-kDa MMP-8 isoform was not detectable. Tear fluid flow-corrected secretion of MT1-MMP was significantly higher in PRK-treated patients. Soluble 80-kDa MT1-MMP immunoreactivities were detected in tears of both healthy control subjects and PRK-treated patients, and may represent a complex captured by tissue inhibitor of metalloproteinase (TIMP)-2. Human PMN pro-MMP-8 was converted to the active form by MT1-MMP, and TIMP-2 prevented this activation., Conclusions: Corneal renewal eventually occurs at a high rate and is affected by the rate of corneal collagen and other matrix protein breakdown. Accordingly, tear fluid MMP-8 and MT1-MMP levels were shown to be constantly high in normal subjects. With PRK, a fast wound-healing process was associated with even higher MMP-8 and MT1-MMP levels and their activation. The results suggest a role for a MMP-8 and MT1-MMP network in the corneal wound-healing cascade. Furthermore, MT1-MMP (MMP-14) seems to activate pro-MMP-8.