Maria Markoulli

TFOS DEWS II pathophysiology report

The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via a Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level across the ocular surface, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjögren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.

The TFOS International Workshop on Contact Lens Discomfort: Report of the contact lens interactions with the ocular surface and adnexa subcommittee

Efron, N., Jones, L., Bron, A. J., Knop, E., Arita, R., Barabino, S., … Markoulli, M. (2013). The TFOS International Workshop on Contact Lens Discomfort: Report of the Contact Lens Interactions With the Ocular Surface and Adnexa Subcommittee. Investigative Opthalmology & Visual Science, 54(11), TFOS98. https://doi.org/10.1167/iovs.13-13187

Validation of the Flush Method as an Alternative to Basal or Reflex Tear Collection

Purpose: To validate the more easily applicable “flush” tear collection technique as a viable alternative to basal and reflex tear collection. Materials and Methods: Total protein content (TPC) and immunoglobulin A (IgA) concentrations were determined in the basal, reflex, and flush tears of 16 healthy non-contact lens wearers. The overall protein profile was established using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Results: Collection-rates were 4.6u2009±u20096.7 μl/min, 13.9u2009±u200911.1 μl/min, and 25.7u2009±u200912.4 μl/min for the basal, reflex, and flush tears, respectively. TPC was 7.14u2009±u20092.22u2009mg/mL, 6.01u2009±u20092.11u2009mg/mL, and 3.79u2009±u20091.51mg/u2009mL for basal, reflex, and flush tears, respectively, with flush tears being significantly less concentrated than basal (pu2009=u20090.001) and reflex (pu2009=u20090.008). IgA concentration was 1.04u2009±u20090.29u2009mg/u2009mL, 0.64u2009±u20090.26u2009mg/mL, and 0.65u2009±u20090.23u2009mg/mL for basal, reflex, and flush tears, respectively, with basal tears being significantly more concentrated (pu2009<u20090.001). As a percentage of TPC, IgA represented 19.8u2009±u200914.9%, 11.4u2009±u20093.9%, and 19.8u2009±u20098.7% for basal, reflex, and flush, respectively. The flush was not significantly different to basal (pu2009=u20091.00) but significantly greater than reflex (pu2009=u20090.02). SDS-PAGE showed the same tear profiles for basal and flush tears. MS identified the most abundant proteins in all tear types. Conclusions: The flush method allows much faster collection than basal secretion sampling but returns essentially the same spectrum of proteins in similar proportions. This behavior is confirmation that the flush technique has utility as a more convenient alternative to basal tear sampling in studies involving composition analysis.

The Diurnal Variation of Matrix Metalloproteinase-9 and Its Associated Factors in Human Tears

PURPOSEnMatrix metalloproteinases (MMPs) are degrading enzymes which maintain and remodel tissue architecture. Upregulation of MMP-9 has been associated with corneal erosions and ulceration. As these conditions are often exacerbated on waking, suggesting that degrading activity is upregulated overnight, this study set out to determine the diurnal variation of MMP-9, Tissue Inhibitor of Metalloproteinase (TIMP)-1, and Neutrophil Gelatinase-Associated Lipocalin (NGAL).nnnMETHODSnFlush tears were collected from 46 healthy, non-contact lens wearers at midday, before sleep, and immediately on waking. Total protein content (TPC) was measured using the bicinchoninic acid method, and MMP-9, TIMP-1, and NGAL concentrations were measured using sandwich enzyme-linked immunoassay. Statistical analysis was performed using repeated measures analysis of variance.nnnRESULTSnTPC was 3.4 ± 1.5 mg/mL, 5.0 ± 3.7 mg/mL and 15.5 ± 8.4 mg/mL for midday, before sleep, and on waking respectively, the latter being significantly greater than the other two (P < 0.001). MMP-9 concentrations at the corresponding time points were 9.8 ± 14.3 ng/mL, 8.5 ± 11.7 ng/mL, and 2000.7 ± 1950.7 ng/mL. Again, the value on waking was significantly greater than the previous two visits (P < 0.001). TIMP-1 concentrations exceeded those of MMP-9 at midday but the ratio of the two reversed on awakening.nnnCONCLUSIONSnConcentrations of MMP-9 are negligible during the day and completely inhibited by TIMP-1. On awakening, MMP-9 increases 200-fold, an increase that is not completely inhibited by TIMP-1. This diurnal change, along with the presence of NGAL which protects MMP-9 from degradation, suggests that the closed eye is an environment conducive to extracellular matrix remodeling.

The Proview Eye Pressure Monitor: influence of clinical factors on accuracy and agreement with the Goldmann tonometer

Purpose:u2002 To investigate (i) the effect of probe position on the lid when using the Proview Eye Pressure Monitor (Proview) and (ii) the accuracy of the Proview and its agreement with the Goldmann applanation tonometer (GAT) in a normal population.

Impact of duration of contact lens wear on the structure and function of the meibomian glands.

To investigate the effects of the duration of contact lens (CL) wear on the meibomian glands (MGs), eyelid and tear film.

Tear film break‐up time in rabbits

Rabbits have a longer inter‐blink time (approximately 10 minutes) compared with humans (five to eight seconds), suggesting that rabbits have a much more stable tear film. Using fluorescein, the tear break‐up time of rabbits has been reported to be similar to that of humans. This study set out to measure the tear break‐up time in rabbits using non‐invasive methods and to establish the pattern of tear break‐up compared to humans.

Lid and conjunctival micro biota during contact lens wear in children.

Purpose. To determine the types of microorganisms and their frequency of isolation on the external ocular surface in children wearing soft contact lenses on a daily wear schedule. Methods. Children aged 8 to 14 years were fitted with commercially available, soft contact lenses which were worn on a daily wear basis. The upper bulbar conjunctiva and the lower lid margins of each eye were swabbed at baseline and then at 6-monthly intervals for 2 years during lens wear. Swabs were processed, cultured and microorganisms identified using standard microbiological techniques. Results. At baseline, 36% of the upper bulbar conjunctivae and 53% of the lower lid margins were contaminated with predominantly gram-positive bacteria. Recovery of samples with positive growth from the upper bulbar conjunctivae decreased during lens wear and ranged from 27 to 14%. The difference was statistically significant at the 12-month visit (14%, p = 0.011). The lower lid margins generally showed similar levels of contamination as at baseline except for less contamination at the 12-month visit (33%) (p = 0.05). Staphylococcus epidermidis and Propionibacterium sp. were the predominant microorganisms isolated from both the sites with and without lens wear. Gram-negative bacteria were infrequent and when isolated, the numbers of colonies were few. Fungus was isolated in a small percent (ranging from 0 to 4.8%) of eyes at various time points. Conclusions. The external ocular surface of children is contaminated with gram-positive bacteria that are resident micro biota. During lens wear, there was a trend for lesser recovery of organisms from the upper bulbar conjunctival samples, but there was no change in the type of microorganisms isolated. At all times, lower lid margins showed consistently greater colonization and is a source of potential contamination of the conjunctival surface.

Effect of contact lens wear on the diurnal profile of matrix metalloproteinase 9 in tears.

Purpose This study set out to determine how contact lens wear affects the profile of matrix metalloproteinase 9 (MMP-9) in the tear film. Methods Flush tears were collected from 47 healthy neophytes before lens wear, during the first day of lens wear, and after 1 month adaptation. Participants were randomized to either Acuvue Oasys or O2OPTIX with the choice of extended (EW) or daily wear (DW). Each time, tears were collected at midday, before sleep, and on waking and analyzed for concentrations of total protein, MMP-9, and its regulators TIMP-1 (tissue inhibitor of metalloproteinases 1) and NGAL (neutrophil gelatinase–associated lipocalin). Results Initial extended contact lens wear resulted in significantly elevated MMP-9 levels on waking (3598.7 ± 3229.1 ng/mL) compared with the same time point at baseline (2123.3 ± 1762.8; p = 0.02), whereas DW remained unchanged (2373.0 ± 2091.6 ng/mL; p = 0.61). After 1 month of EW, the levels on awakening were no longer different to those of baseline (2408.2 ± 1376.1 ng/mL; p = 0.63). The MMP:TIMP ratio during EW was greater after the first night (18.6 ± 19.9) than both no wear (13.2 ± 13.4) and 1 month (10.4 ± 7.7), but only the latter was significant (p = 0.048). The profile of NGAL did not differ from baseline (p = 0.055). Conclusions In the neophyte, the initial period of overnight lens wear seems to disturb the tear film homeostasis, as indicated by a significant increase in MMP-9 on awakening. The return to baseline by 1 month suggests that an adaptive process takes place. No comparable changes are seen in DW.

Divalent cations in tears, and their influence on tear film stability in humans and rabbits

PURPOSEnReduced tear film stability is reported to contribute to dry eye. Rabbits are known to have a more stable tear film than humans. Thus, we sought to examine the tears of rabbits and humans for metal cations, and to test how they influence tear film stability.nnnMETHODSnTears were collected from 10 healthy humans and 6 rabbits. Tear osmolality was measured by vapor pressure osmometer, and metals analyzed using inductively coupled plasma (ICP) mass spectrometry or ICP atomic emission spectroscopy. The influence of divalent cations on tears was analyzed by measuring surface tension using the Langmuir trough in vitro, using different concentrations of cations in the subphase, and grading the tear break-up in rabbits in vivo after instillation of chelating agents.nnnRESULTSnRabbit tears had a higher osmolality compared to humans. Major metals did not differ between species; however, rabbits had higher levels of Mg(2+) (1.13 vs. 0.39 mM) and Ca(2+) (0.75 vs. 0.36 mM). In rabbit tears in vitro, diminishing divalent cations resulted in a decrease in the maximum surface pressure from 37 to 30 mN/m. In vivo, an increase in the amount of tear film that was broken-up was found. In contrast, when changing divalent cation concentrations in human tears, the maximum surface pressure remained at 26 mN/m.nnnCONCLUSIONSnThe normal osmolality of rabbit tears is significantly higher than that in humans. While divalent cations had little influence on human tears, they appear to have an important role in maintaining tear film stability in rabbits.