Sruthi Srinivasan

Infrared imaging of meibomian gland structure using a novel keratograph.

Purpose. To examine the ability of a novel non-contact device (Keratograph 4) to image the meibomian gland (MG) structures and their morphological changes in the upper and lower eyelids. Methods. Thirty-seven participants (mean age 57.8 ± 8.5 years; 3 males and 34 females) completed the Ocular Surface Disease Index questionnaire to assess dryness symptoms. Meibum secretion quality score, number of blocked gland orifices, and meibum expressibility scores were assessed. The lower lid (LL) and upper lid (UL) of all subjects were everted and images of the MGs were taken using the Keratograph 4 (OCULUS). A MG dropout score (MGDS) due to complete or partial gland loss of both lids was obtained using a subjective 4-grade scoring system, and digital analysis of the images using ImageJ was performed. Presence of tortuosity and visible acinar changes of the MGs were also noted. Results. MGDS for both lids was significantly positively correlated with the Ocular Surface Disease Index score (r = 0.51; p < 0.05). The MGDS determined using the digital grading was also significantly positively correlated (UL: r = 0.68, p < 0.05; LL: r = 0.42, p < 0.05). The sum of the MGDS for both lids using the subjective grading scale was significantly different between the non-MGD and MGD group (1.3 ± 1.0 vs. 3.1 ± 1.1; p = 0.0004). MGDS assessment using the digital grading was significantly different between non-MGD (UL = 6%, LL = 8%) and MGD group (UL = 32%, LL = 42%; p = 0.001). Tortuous MG was observed only on the UL in 6% of the participants. Visible acinar changes were noted in 40% of the study participants. Conclusions. Infrared meibography is now possible in a clinical setting using commercially available devices, and meibography can help determine differences in MG structure in subjects symptomatic of dry eye.

iTRAQ Quantitative Proteomics in the Analysis of Tears in Dry Eye Patients

PURPOSE We analyzed the change in protein expression of tear film proteins in dry eye (DE) and non-DE (NDE) patients using isobaric tag for relative and absolute quantitation (iTRAQ) technology. METHODS We categorized 24 participants into NDE, and mild (MDE), moderate-to-severe (MSDE), and mixed (MXDE) DE on the basis of clinical DE tests. Tear samples (n = 6 subjects/group) were collected using Schirmers strips. Proteins were extracted from strips and were quantified using the Bradford assay. Protein from each sample was pooled as internal standard (IS), and 20 μg protein from each sample and the IS were digested and labeled with different tandem mass tag (TMT) isobaric mass tag labeling reagent. The reaction was quenched and the labeled peptides were mixed. Samples were injected for liquid chromatography-mass spectrometry (LC/MS/MS) analysis on the Orbitrap mass spectrometer. Bioinformatic analyses were performed using protein information resource (PIR). RESULTS Combined results showed a total of 386 proteins in tears as determined by the iTRAQ experiments. An average of 163 proteins was detected in each of 6 biologic replicates. Of those, 55% were detected 6 times and 90% were detected multiple times (>2). In addition to the down-regulation of commonly reported proteins, such as lipocalin-1, lysozyme, and prolactin-inducible protein across all sub groups of DE, a number of proteins were significantly differentially regulated in MSDE and other subgroups of DE. A greater number of proteins were down-regulated in MSDE versus MDE, and the specific functions involved include response to stimulus (8 vs. 6 proteins), immune system process (6 vs. 4), regulation of biologic processes (3 vs. 3), and ion transport (2 vs. 2). CONCLUSIONS iTRAQ is one of the newest tools for quantitative mass spectrometry in tear proteome research. Differences in the protein ratios can be detected between normal and DE patients. PIR is a useful resource to interpret pathways and functions of proteins.

Molecular Structure of Interfacial Human Meibum Films

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

Tear osmolality and ferning patterns in postmenopausal women.

Purpose. To compare tear osmolality and ferning patterns in postmenopausal women (PMW) with and without dry eye symptoms. Methods. Thirty-seven healthy PMW (>50 years of age), not on hormone replacement therapy, were categorized as being symptomatic or asymptomatic of dry eye based on their responses to an Allergan “Single-Item Score Dry Eye Questionnaire” (SIDEQ). They subsequently completed the Allergan “Ocular Surface Disease Index” (OSDI) questionnaire. Tear samples were collected from participants to evaluate osmolality and ferning patterns. A novel freezing point depression osmometer (Advanced Instruments Inc., Model 3100 Tear Osmometer), was used to measure the osmolality of the tear film. The tear ferning test was performed and evaluated for the quality of ferning based on the Rolando grading system. Results. SIDEQ responses revealed 21 symptomatic and 16 asymptomatic participants. The OSDI total score was 6.5 ± 5.9 for the non–dry-eyed (NDE) group and 25.7 ± 12.4 for the dry-eyed (DE) group. The subscores for the DE group were significantly greater than the NDE group (p < 0.001). Osmolality values in DE individuals were significantly different from NDE (328.1 ± 20.8 vs. 315.1 ± 11.3 mOsm/kg; p = 0.02). Fifty percent of the DE participants showed type II ferning patterns and 29% of the DE participants showed type III ferning patterns, whereas the NDE participants showed either type I (44%) or II (66%) ferning patterns. There was a significant difference between the DE and NDE participants for the ferning patterns (p = 0.019). There was no significant correlation between tear osmolality and tear ferning (DE: r = 0.12; p > 0.05, NDE: r = −0.17; p > 0.05). Conclusions. Osmolality in mild and moderately DE PMW is higher than in NDE PMW and tear ferning is a rapid, simple, noninvasive laboratory procedure that indicates altered tear quality in PMW with symptoms of dry eye.

Protein deposition and clinical symptoms in daily wear of etafilcon lenses.

Purpose. To determine the relationship between clinical signs and symptoms and protein deposition over 8 h of wear of etafilcon A lenses in symptomatic and asymptomatic contact lens wearers. Methods. Thirty adapted soft contact lens wearers (16 symptomatic and 14 asymptomatic) were fitted with etafilcon A lenses. In vivo wettability, non-invasive tear break-up time, and subjective symptoms (vision, comfort, and dryness) were assessed at baseline and after 2, 4, 6, and 8 h. After 2, 4, 6, and 8 h time points, lenses were collected, and total protein, total lysozyme, and active lysozyme deposition were assessed. Results. There was a significant reduction (p = 0.032) in the non-invasive tear break-up time at 8 h in both groups. In the symptomatic group, there was a significant reduction in subjective comfort and dryness ratings at 6 and 8 h measurement with respect to baseline (p < 0.05). There was a significant increase in total lysozyme and total protein deposition (p = 0.027) across all time points in both groups; most of the lysozyme remained active (>94% at 8 h). Pearsons correlations between subjective symptoms and protein deposition showed poor correlations for total protein/lysozyme and any subjective factor (r < 0.3; p > 0.05), and only weak correlations between dryness and % active lysozyme (r = 0.3 to 0.5 for all time points). However, stronger correlations were found between active lysozyme and subjective comfort (r = 0.6 to 0.7; p < 0.001). Conclusions. In addition to investigating total protein deposited on contact lenses, it is of significant clinical relevance to determine the conformational state of the deposited protein.

Quantification of non-polar lipid deposits on senofilcon a contact lenses.

Purpose. To quantify non-polar lipids deposited on senofilcon A silicone hydrogel contact lenses (J&J Acuvue OASYS) when disinfected with a no-rub one-step hydrogen peroxide system (CIBA Vision ClearCare) and a care system preserved with Polyquad & Aldox (Alcon OPTI-FREE RepleniSH). Methods. Thirty existing soft lens wearers symptomatic of dryness were enrolled into a 4-week prospective, randomized, bilateral eye (lens type), cross-over (care regimen), daily wear, double masked study. Subjects were refitted with senofilcon A lenses, which were replaced biweekly. During each period of wear, participants used either the peroxide or preserved system. After each period of wear, lenses were collected and lipid was extracted using 1.5 ml of a 2:1 chloroform:methanol solution for 3 h at 37°C. Lens extracts were analyzed for non-polar lipids [cholesterol oleate (CO), cholesterol, oleic acid (OA), triolein, and OA methyl ester] using normal phase high-performance liquid chromatography. Results. The total lipid (sum of CO and cholesterol) detected was 34 ± 28 &mgr;g/lens for the peroxide-based system and 22 ± 21 &mgr;g/lens for the system preserved with Polyquad and Aldox (p = 0.029). Although there was no difference between products for cholesterol (1.4 vs. 1.3 &mgr;g/lens; p = 0.50), use of a system preserved with Polyquad and Aldox resulted in significantly less deposited CO (33 ± 28 vs. 21 ± 20 &mgr;g/lens; p = 0.033). Approximately, 95% of the detectable lipid deposited on the material was CO, followed by cholesterol. OA and triolein contributed <1% of the total lipid and no OA methyl ester was found on any of the lenses. Conclusions. A care system preserved with Polyquad and Aldox removed higher amounts of CO from senofilcon A contact lenses used for 2 weeks than a peroxide-based system, in soft lens wearers who were symptomatic of dry eye.

Apparent time-dependent differences in inferior tear meniscus height in human subjects with mild dry eye symptoms

Purpose:  The aim of the study was to track the volume of tears contained in the inferior tear meniscus over the course of the day in subjects with symptoms of mild dry eye and a control asymptomatic group.

Repeatability of grading meibomian gland dropout using two infrared systems.

Purpose To determine the interobserver and intraobserver repeatability in using the OCULUS Keratograph 4 (K4) and 5M (K5M) to grade meibomian gland (MG) dropout using meibography grading scales. Methods The inferior and superior eyelids of 40 participants (35 women, 5 men; mean age = 32 years) were imaged three times each on both instruments. The images were split into one training and two study sets; the latter were graded (four-point meibography scale) by two observers on two separate occasions (24 hours apart) to determine repeatability. Semiobjective quantification of percentage MG dropout was conducted using ImageJ on K4 and K5M images. A finer seven-point meibography scale was used to grade a separate set of K5M images. Results For the four-point scale, interobserver mean difference (MD) (±SD) was 0.08 (±0.55) on day 1 and 0.13 (±0.50) on day 2, and the concordance correlation coefficient (CCC) was 0.79 and 0.81 on days 1 and 2, respectively. Intraobserver MD (±SD) was 0.04 (±0.54), CCC = 0.79 for observer 1; intraobserver MD (±SD) was −0.09 (±0.60), CCC = 0.74 for observer 2. For the seven-point scale, interobserver MD (±SD) was 0.05 (±0.45), CCC = 0.89 on day 1, and interobserver MD (±SD) was 0.01 (±0.41), CCC = 0.91 on day 2. Intraobserver MD (±SD) was −0.10 (±0.35), CCC = 0.93 for observer 1, and intraobserver MD (±SD) was −0.06 (±0.30), CCC = 0.95 for observer 2. Percentage dropout measured between the K4 and K5M images showed lack of agreement, with 21.8% coefficient of repeatability. There was no significant correlation (r < 0.2; p > 0.05) between meibography score and clinical signs (corneal staining, gland expressibility, telangiectasia, vascularity, lash loss); however, there was a high correlation (r = 0.77; p < 0.05) between meibography score with percentage dropout. Conclusions Observers graded from −1 to +1 grade units between and within themselves for a four-point scale, 95% of the time. Although the interobserver and intraobserver repeatability of the K4 and K5M were very similar, a high rate of disagreement in percentage dropout between K4 and K5M images suggests that the two instruments cannot be interchanged. Meibomian gland dropout scores did not correlate significantly with clinical signs. Using a finer scale may be beneficial for detecting change.

The TFOS International Workshop on Contact Lens Discomfort: report of the management and therapy subcommittee.

Erratum in Invest Ophthalmol Vis Sci. 2014 Sep;55(9):5588. Miller, William S [coorected to Miller, William L].

Infrared Imaging of Meibomian Glands and Evaluation of the Lipid Layer in Sjogren's Syndrome Patients and Nondry Eye Controls

PURPOSE The purpose of this study was to evaluate meibomian gland dropout and lipid layer thickness (LLT) in patients with and without Sjögrens syndrome dry eye (SS). METHODS We recruited 11 participants with SS (males/females [M/F], 1:10; mean age = 56.0 ± 9.1 years) and 10 control subjects without dry eye (M/F, 3:7; mean age = 58.5 ± 4.7 years). All participants completed the Ocular Surface Disease Index (OSDI) questionnaire. The LLT was assessed using the Tearscope Plus based on the appearance of the lipid layer. Noninvasive tear break-up time (NITBUT) also was measured. The lower and upper lids were everted, and the meibomian glands were imaged using the infrared camera of the Keratograph 4. A meibomian gland dropout score due to gland loss was obtained. Statistical analysis was conducted using the Mann-Whitney U test and correlations were determined using Spearman rank correlations. RESULTS Of the SS participants, 100% reported ocular and oral dryness symptoms in the AECC questionnaire. The SS group recorded a higher OSDI score (median = 48.00, interquartile range [IQR] 23.0-56.2 vs. 2.1, IQR 0.0-2.6; P < 0.001), reduced LLT (median [IQR] = 15.0 [15.0-15.0] vs. 60.0 [45.0-100.0] nm; P = 0.001), and lower NITBUT (median [IQR] = 3.7 [2.5-4.2] vs. 9.5 [6.4-17.6] sec; P < 0.001) compared to the controls. Digital meibomian gland dropout score (% dropout) was significantly higher for the SS group (16.0% [IQR 12.1-40.0%] vs. 6.7% [IQR 1.5-12.7%]; P = 0.01). Subjective meibomian gland dropout score (0-6 score) was significantly higher for the SS group (median [IQR] = 1.5 [1.0-4.0] vs. 1.0 [0.0-1.25]; P = 0.03). CONCLUSIONS Patients with SS showed higher meibomian gland dropout scores and reduced LLT and NITBUT, which likely contribute to the severe dry eye symptoms reported by SS subjects.