Proteomics analyses of human optic nerve head astrocytes following biomechanical strain.

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor ß1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.

In vitro contact angle analysis and physical properties of blister pack solutions of daily disposable contact lenses.

OBJECTIVES: The purpose of this study was to measure the advancing and receding contact angles (CAs) of five daily disposable (DD) lenses and the osmolality, surface tension (ST), and pH of each blister pack solution. METHODS: The advancing and receding CAs were measured directly out of the blister pack for five DD lenses: omafilcon A (CooperVision), nelfilcon A (CIBA Vision), modified nelfilcon A (CIBA Vision), etafilcon A (Johnson & Johnson), and narafilcon A (Johnson & Johnson). Advancing CAs were measured using sessile drop and Wilhelmy balance methods. Receding CAs were measured using the Wilhelmy balance method. ST, pH, and osmolality were measured for each blister pack solution from all the DD lenses. RESULTS: The advancing CAs for the nelfilcon A lenses were statistically lower (P<0.05) than the advancing CAs of the other three lenses. The receding CAs for etafilcon A were statistically lower (P<0.05) than the receding CAs for the other four lenses. The pH of all the blister pack solutions was relatively neutral (7.01-7.43). The ST of the blister pack solution for the modified nelfilcon A was significantly lower (P<0.05) than the ST of all other blister pack solutions. The osmolality of the blister pack solutions for the etafilcon A and narafilcon A lenses were significantly higher (P<0.05) than the other blister pack solutions. CONCLUSIONS: The modified nelfilcon A lens had low advancing CAs and low receding CAs showing minimal hysteresis. The blister pack solution for the modified nelfilcon A lens had the lowest ST, a low osmolality, and a neutral pH.

Proteomics analyses of activated human optic nerve head lamina cribrosa cells following biomechanical strain.

PURPOSE: To determine protein regulation following activation of human, optic nerve head (ONH), lamina cribrosa (LC) cells in response to mechanical strain. METHODS: LC cells were isolated and grown from donor tissue in specific media at 37°C and 5% CO(2) humidified incubator. Cells were grown to confluence on collagen I-coated flexible-bottom culture plates, rinsed with Dulbecco's phosphate-buffered saline, and left for 24 hours in serum-free media. They were subjected to 3% or 12% cyclic equiaxial stretch for 2 or 24 hours using a commercial strain-unit system. Control cells were serum-deprived and incubated without stretch for 24 hours. Nano liquid chromatography-mass spectrometry analysis with isobaric tags for relative and absolute quantitation labeling was used to determine protein regulation. RESULTS: In all, 526 proteins were discovered at a 95% confidence limit. Analysis of associated pathways and functional annotation indicated that the LC cells reacted in vitro to mechanical strain by activating pathways involved in protein synthesis, cellular movement, cell-to-cell signaling, and inflammation. These pathways indicated consistent major protein hubs across all stretch/time conditions involving transforming growth factor-ß1 (TGFß1), tumor necrosis factor (TNF), caspase-3 (CASP3), and tumor protein-p53 (p53). Among proteins of particular interest, also found in multiple stretch/time conditions, were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66), and eukaryotic translation initiation factor 5A (eIF-5A). CONCLUSIONS: Pathway analysis identified major protein hubs (TGFß1, TNF, CASP3, p53) and pathways all previously implicated in cellular activation and in the pathogenesis of glaucomatous optic neuropathy. Several specific proteins of interest (BAG5, NO66, eIF-5A) were identified for future investigation as to their role in ONH glial activation.

Physical properties of soft contact lens solutions.

PURPOSE: To investigate the physical properties of commercially available soft contact lens solutions. METHODS: The pH, osmolality, surface tension (ST), and viscosity of various soft contact lens solutions were measured at room temperature. Viscosity measurements were also taken at 34 degrees C. The solutions examined were Opti-Free Express (OFX), Opti-Free RepleniSH (OFR), Complete Moisture Plus (COM), UltraCare (UC), ReNu MultiPlus, Sensitive Eyes, AOSept (AO), Clear Care, SoloCare Aqua, and SoftWear saline. The peroxide solutions were measured before and after neutralization. RESULTS: The pH of most solutions was close to neutral (range 7.00-7.36), except for OFX and neutralized AO and Clear Care. The osmolality values of most solutions were in the 275 to 310 mOsm/kg range. OFX exhibited a significantly lower osmolality (225 mOsm/kg; p < 0.001), whereas UC was significantly higher (329 mOsm/kg; p < 0.001). Neutralized AO and SoftWear saline had ST values of approximately 67 mN/m. OFX, OFR, and SoloCare Aqua exhibited low ST values in the 30 to 35 mN/m range. The remaining solutions exhibited intermediate ST values of approximately 40 mN/m. These three groupings were significantly different (p < 0.001). The average viscosity of most solutions at room temperature was between 0.95 and 1.26 cP, except for COM (3.02 cP; p < 0.001). At 34 degrees C, the average viscosity of most solutions was between 0.70 and 0.83 cP, except for COM, which had a viscosity of 1.92 cP (p < 0.001). The un-neutralized peroxide solutions had very different pH and osmolality values from all the solutions that would directly contact the eye (p < 0.001). Their viscosity and ST values were similar (p = NS). CONCLUSIONS: This study detailed many physical properties of soft lens solutions that are not readily available and indicated that certain properties vary significantly among these products.

The impact of lipid on contact angle wettability.

PURPOSE: To analyze the effect of in vitro lipid doping on conventional hydrogel (CH) and silicone hydrogel (SH) lens wettability, assessed by sessile drop contact angle (CA) measurement. METHODS: Nine contact lens materials, five SHs and four CH, were incubated with two different lipid tear solutions (LTS) containing cholesterol, cholesteryl oleate, oleic acid, oleic acid methyl ester, and triolein. The first LTS was a "low" concentration solution, which was close to human values, and the second was a "high" concentration. Lenses were soaked in the two LTS types for 2 or 5 days and compared with lenses soaked in phosphate buffered saline (PBS) only. After soaking, advancing CAs were measured on a customized computerized device using a sessile drop method. RESULTS: Compared with PBS, CAs for untreated SHs were unaffected by soaking in the LTS, with typical CA values of >95 degrees (p > 0.05). The surface-treated SH materials exhibited markedly reduced CAs after lipid exposure, with the high concentration LTS reducing the CA to <5 degrees (p < 0.01). The CH materials all exhibited lower CAs after soaking, with values typically decreasing to 35 degrees , which was significantly lower than that seen with PBS (p < 0.01). CONCLUSION: Exposure to lipid may improve the wettability of certain SH and CH materials, particularly those SH materials that are surface treated. This may help to explain why certain SH materials appear to improve in comfort for some patients during the first few hours or days of wear.