Occurrence of corneal sub-epithelial microneuromas and axonal swelling in people with diabetes with and without (painful) diabetic neuropathy.

AIMS/HYPOTHESIS: Non-invasive in vivo corneal confocal microscopy is gaining ground as an alternative to skin punch biopsy to evaluate small-diameter nerve fibre characteristics. This study aimed to further explore corneal nerve fibre pathology in diabetic neuropathy. METHODS: This cross-sectional study quantified and compared corneal nerve morphology and microneuromas in participants without diabetes (n=27), participants with diabetes but without distal symmetrical polyneuropathy (DSPN; n=33), participants with non-painful DSPN (n=25) and participants with painful DSPN (n=18). Clinical and electrodiagnostic criteria were used to diagnose DSPN. ANCOVA was used to compare nerve fibre morphology in the central cornea and inferior whorl, and the number of corneal sub-epithelial microneuromas between groups. Fisher's exact tests were used to compare the type and presence of corneal sub-epithelial microneuromas and axonal swelling between groups. RESULTS: Various corneal nerve morphology metrics, such as corneal nerve fibre length and density, showed a progressive decline across the groups (p<0.001). In addition, axonal swelling was present more frequently (p=0.018) and in higher numbers (p=0.03) in participants with painful compared with non-painful DSPN. The frequency of axonal distension, a type of microneuroma, was increased in participants with painful and non-painful DSPN compared to participants with diabetes but without DSPN and participants without diabetes (all p≤0.042). The combined presence of all microneuromas and axonal swelling was increased in participants with painful DSPN compared with all other groups (p≤0.026). CONCLUSIONS/INTERPRETATION: Microneuromas and axonal swelling in the cornea increase in prevalence from participants with diabetes to participants with non-painful DSPN and participants with painful DSPN.

Corneal Dendritic Cell Dynamics Are Associated with Clinical Factors in Type 1 Diabetes.

Time-lapsed in vivo corneal confocal microscopy (IVCCM) has shown that corneal dendritic cells (DCs) migrate at approximately 1 µm/min in healthy humans. We have undertaken IVCCM of the whorl region to compare the density of rounded DCs, and DCs with (wDCs) and without (woDCs) dendrites and dynamics; trajectory (length travelled/time), displacement (distance from origin to endpoint/time) speeds and persistence ratio (displacement/trajectory) of woDCs in subjects with type 1 diabetes (T1D) (n = 20) and healthy controls (n = 10). Only the wDC density was higher (p = 0.02) in subjects with T1D compared to controls. There was no significant difference in cell dynamics between subjects with T1D and controls. woDC density correlated directly with HDL cholesterol (r = 0.59, p = 0.007) and inversely with triglycerides (r = −0.61, p = 0.005), whilst round-shaped cell density correlated inversely with HDL cholesterol (r = −0.54, p = 0.007). Displacement, trajectory, and persistency correlated significantly with eGFR (mL/min) (r = 0.74, p < 0.001; r = 0.48, p = 0.031; r = 0.58, p = 0.008, respectively). We show an increase in wDC density but no change in any other DC sub-type or alteration in cell dynamics in T1D. However, there were associations between DC density and lipid parameters and between DC dynamics and renal function. IVCCM provides evidence of a link between immune cell dynamics with lipid levels and renal function.

Lid Margin Score Is the Strongest Predictor of Meibomian Area Loss.

PURPOSE: Although meibography provides direct evidence of gland dropout in meibomian gland dysfunction, this specialized technique is not available in most clinics. The primary aim was to determine which clinical ocular marker was most related to meibomian area loss. A secondary aim was to determine associations with confocal microscopy imaging of the lid margin. METHODS: One hundred participants from age 18 to 65 years were recruited. Measurements of the right eye and its upper eyelid, where relevant, included noninvasive tear break-up time, bulbar and limbal redness scores, blepharitis score, lipid layer thickness, number of parallel conjunctival folds, tear osmolarity, corneal fluorescein staining, phenol red thread test, lid margin score, meibography, and in vivo confocal microscopy. Participants also completed the Ocular Surface Disease Index questionnaire. The relationships between the measurements were determined using the Spearman correlation. The receiver operating characteristic curve and area under the receiver operating characteristic curve were used to determine the cutoff value of clinical markers. RESULTS: Significant correlations were found between meibomian area and lid margin score (r = -0.47, P < 0.01), and meibomian tortuosity and lid signs of blepharitis (r = -0.32, P < 0.01). Area under the receiver operating characteristic curve analysis revealed that a lid margin score of ≥1.70 detected meibomian area loss with a sensitivity of 0.58 and a specificity of 0.86. There were significant correlations between meibomian area and orifice area at 30 µm depth (r = -0.25, P = 0.01). CONCLUSIONS: The lid margin score was most related to the meibomian area and thus the best predictor of undiagnosed meibomian area loss.

In vivo immune cell dynamics in the human cornea.

In vivo confocal microscopy (IVCM) allows the evaluation of the living human cornea at the cellular level. The non-invasive nature of this technique longitudinal, repeated examinations of the same tissue over time. Image analysis of two-dimensional time-lapse sequences of presumed immune cells with and without visible dendrites at the corneal sub-basal nerve plexus in the eyes of healthy individuals was performed. We demonstrated evidence that cells without visible dendrites are highly dynamic and move rapidly in the axial directions. A number of dynamic cells were observed and measured from three eyes of different individuals. The total average displacement and trajectory speeds of three cells without visible dendrites (N = 9) was calculated to be 1.12 ± 0.21 and 1.35 ± 0.17 µm per minute, respectively. One cell with visible dendrites per cornea was also analysed. Tracking dendritic cell dynamics in vivo has the potential to significantly advance the understanding of the human immune adaptive and innate systems. The ability to observe and quantify migration rates of immune cells in vivo is likely to reveal previously unknown insights into corneal and general pathophysiology and may serve as an effective indicator of cellular responses to intervention therapies.

Association between conjunctival goblet cells and corneal resident dendritic cell density changes in new contact lens wearers.

BACKGROUND: To explore the interlink between conjunctival goblet and corneal dendritic cell density after six months of lens wear and to predict dendritic cell migration to the central cornea based on goblet cell loss in the conjunctiva as a response to contact lens wear. METHODS: Sixty-nine subjects who had never previously worn contact lenses were observed for six months; 46 were fitted with contact lenses and 21 served as a control group. Corneal confocal microscopy was used to quantify goblet and dendritic cell density before and after six months of daily lens wear. Symptomatic and asymptomatic groups were identified in the lens-wearing group using a combination of signs and symptoms present. Pearson's correlation was used to determine associations between the total change of cell densities after six months of lens wear. RESULTS: At baseline, there was no association between conjunctival goblet and corneal dendritic cell density (p > 0.05). After six months, there was an inverse association between the absolute change of conjunctival goblet and corneal dendritic cell density (ρ = -0.34, p = 0.03) in all participants (n = 69). Dendritic cell density in the central cornea was increased by 1.5 cells/mm2 for every decrease of 1 goblet cell/mm2 in the conjunctiva. CONCLUSIONS: After six months of wear, contact lens-induced goblet cell loss can partially predict resident corneal dendritic cell migration to the central cornea (observed as an increase in dendritic cell density). The associations between total cell density change after six months was established in wearers regardless of lens symptomatology, suggesting that cell density changes as a physiological adaptation to regulate the effect of contact lens wear on the ocular surface.

Development of Feasible Methods to Image the Eyelid Margin Using In Vivo Confocal Microscopy.

PURPOSE: To develop a feasible method to image eyelid margin structures using in vivo confocal microscopy (IVCM) for use in clinical research. Second, to assess the association between IVCM and meibography images. METHODS: IVCM was performed on the central upper eyelid margin of 13 healthy participants (31 ± 5 years). Overall morphology montages (1600 × 1600 µm) were created of 3 participants. Single frames (400 × 400 µm) of 10 participants were imaged to determine the feasibility of measuring eyelid features. Meibography was performed with EASYTEARview+ in the same 10 participants. ImageJ software was used to quantify image structures. RESULTS: In the montages, structures of rete ridges, meibomian gland openings, and the lid wiper region were observed. The maximum possible montage size, using multiple single frames, was approximately 5200 × 1500 × 150 µm in the X, Y, and Z directions, respectively. The mean number, density, area, perimeter, and shortest and longest diameters of rete ridges of the 9 nonoverlapped frames were 12 ± 2/frame, 73 ± 5/mm, 2504 ± 403 µm, 250 ± 33 µm, 40 ± 6 µm, and 84 ± 13 µm, respectively. Sampling analysis determined at least 5 nonoverlapped frames were necessary to accurately represent the parameters of the ridges. The mean areas of 3 meibomian openings were 785 ± 784 µm, 1036 ± 963 µm, 950 ± 1071 µm, 848 ± 954 µm, 737 ± 831 µm, 735 ± 743 µm, and from 30 µm to 130 µm at 20-µm depth intervals, respectively. No significant association between IVCM and meibography parameters (P = 0.53) was found. CONCLUSIONS: Imaging rete ridges with IVCM should include at least 5 nonoverlapping single frames in the upper eyelid margin. At least 3 openings imaged between 30 and 130 µm at 20-µm depth intervals are recommended to determine the opening area.

The Relationship between Corneal Nerve Morphology and Inflammatory Mediators and Neuropeptides in Healthy Individuals.

SIGNIFICANCE: This study set out to explore the relationship between the ocular surface immune and nervous systems by exploring corneal nerve structure and the presence of inflammatory mediators and neuropeptides in the tear film. PURPOSE: The purpose of this study was to determine the association between corneal nerve morphology and tear film inflammatory mediators and a neuropeptide in healthy individuals. METHODS: Flush tears were collected from both eyes of 21 healthy participants aged 39.7 ± 9.9 years (10 females, 11 males) and analyzed for substance P, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), tumor necrosis factor α, and interleukin 6. In vivo central corneal confocal microscopy was performed on the right eye, and eight images were captured. Variables measured were corneal nerve fiber length (CNFL), corneal nerve density (CNFD), corneal nerve branch density, fiber total branch density, corneal nerve fiber area, corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrac). For each eye, the average across the images and the maximum and minimum values were determined for each variable. Pearson correlation analysis was performed to test for associations. RESULTS: Substance P correlated with CNFrac (max) (r = -0.48, P = .03) and CNFW (min) (r = -0.52, P = .02). TIMP-1 correlated with CNFD (average) (r = -0.53, P = .03), CNFL (average) (r = -0.49, P = .05), CNFrac (max) (r = -0.49, P = .05), and CNFD (min) (r = -0.55, P = .02). Interleukin 6 correlated with CNFW (average) (r = -0.49, P = .05), the standard deviation of CNFL (r = -0.51, P = .04), CNFL (max) (r = -0.50, P = .04), CNFrac (max) (r = -0.50, P = .04), and CNFW (min) (r = -0.55, P = .02). Tumor necrosis factor α, matrix metalloproteinase-9, and its ratio with TIMP-1 did not correlate with any corneal nerve parameters. CONCLUSIONS: Both inflammatory mediators and neuropeptides correlated with measures of corneal nerve morphology, supporting the link between the inflammatory and nervous systems.

Associations between the menstrual cycle, lifestyle factors and clinical assessment of the ocular surface: a prospective observational study.

BACKGROUND: Little is known about the ocular surface changes over the menstrual cycle in young women and the interactions with lifestyle factors. Therefore, the purpose of this study was to explore the associations between modifiable lifestyle factors and menstrual cycle phases on the ocular signs and symptoms of dry eye in young healthy women. METHODS: This was a prospective 1-month observational study. Thirty young healthy women with regular, 24 to 32-day menstrual cycles were recruited. Participants attended three visits at day 7, 14, and 21 (± 1) of their menstrual cycle. At baseline, general health questionnaire was conducted. At each visit, symptomology was quantified using Ocular Surface Disease Index (OSDI) and overall ocular comfort (OOC, visual analogue scale). Ocular signs were assessed using Efron scales, tear break-up time (TBUT) and phenol red thread (PRT). Pearson's correlation was used to determine associations between variables at each visit. RESULTS: A total of 26 participants (mean age = 22.3 ± 3.7 years) with an average menstrual cycle of 28.3 ± 1.3 days completed the 3 visits. The interaction between signs/symptoms and lifestyle factors changed over the cycle. At the follicular phase (day 7), lifestyle factors such diet and levels of stress were correlated with PRT and OSDI, (r = - 0.4, p = 0.022; r = 0.4, p = 0.045 respectively). At the ovulation phase (day 14), the general health score was correlated with OOC scores (r = 0.4, p = 0.047). At day 14, exercise frequency correlated with PRT (r = - 0.4, p = 0.028) and caffeine intake was positively correlate with both; TBUT (r = 0.5, p = 0.020) and PRT (r = 0.5, p = 0.014). At the luteal phase (day 21), we found no correlations between lifestyle factors and dry eye signs or symptoms. CONCLUSIONS: The associations between lifestyle factors and objective and subjective ocular surface assessment appeared to be more pronounced during the ovulation phase of the menstrual cycle compared to the follicular and luteal phases. Misalignment of these factors with the ocular health during the luteal phase could be attributed to central sensitization and changes in levels of luteinising hormone. Natural hormonal changes during menstrual cycle should be considered for diagnosis and treatment of dry eye in young healthy women.

The impact of uncorrected astigmatism on night driving performance.

PURPOSE: To investigate the effect of uncorrected astigmatism on night driving performance on a closed-road circuit. METHODS: Participants included 10 drivers (mean age 24.4 ± 7.0 years), with low to moderate bilateral astigmatism (0.75-1.50 DC), who were regular contact lens (CL) wearers. Vision and night driving performance were assessed in a cross-over design with a toric CL and a best-sphere spherical CL. Binocular visual function measures included photopic and mesopic visual acuity (VA), contrast sensitivity (CS), mesopic motion sensitivity and glare tests (Mesotest® II and Halometer). Night-time driving performance was assessed on a closed-road circuit, which included measures of sign recognition, hazard detection and avoidance, pedestrian recognition distances, lane keeping, speed and overall driving score. RESULTS: Correction of astigmatism with toric CL significantly improved mesopic VA, photopic and mesopic CS, mesopic motion sensitivity, and reduced glare (p < 0.05), compared to the spherical CL; there were no significant effects of visual correction type on photopic VA. Correction of astigmatism using toric CL resulted in significant improvements in night driving performance, compared to driving with spherical CL, particularly for sign recognition, avoidance of low contrast hazards, increased pedestrian recognition distances and overall driving score (p < 0.05). CONCLUSIONS: Correction of low to moderate levels of astigmatism had significant positive effects on night-time driving performance and related tests of visual performance. This has important implications for optical corrections to improve night road safety of drivers with astigmatism.

The Relationship Between Corneal Dendritic Cells, Corneal Nerve Morphology and Tear Inflammatory Mediators and Neuropeptides in Healthy Individuals.

Purpose: To determine the association between corneal dendritic cell (DC) density and corneal nerve morphology and tear film inflammatory mediators and neuromediators in healthy individuals. Methods: Flush tears were collected from 21 healthy participants aged 39.7 ± 9.9 years and analyzed for total protein content (TPC), substance P, matrix-metalloproteinase-9 (MMP-9), tissue inhibitor of MMPs-1 (TIMP-1), tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6). In vivo confocal microscopy was used to assess DC density and corneal nerve morphology. Corneal nerve variables measured were corneal nerve fiber length (CNFL), fiber density (CNFD), branch density (CNBD), fiber total branch density (CTBD), fiber area (CNFA), fiber width (CNFW) and fractal dimension (CNFrac). Results: Participants with DC density over 50 cells/mm2 correlated with CNBD-average (r = 0.7, p = 0.02), CNBD-high (r = 0.6, p = 0.02), CNBD-low (r = 0.6, p = 0.02) CTBD-average (r = 0.7, p = 0.01), CTBD-high (r = 0.6, p = 0.03), CTBD-low (r = 0.7, p = 0.01), CNFA-average (r = 0.7, p = 0.00), CNFA-high (r = 0.7, p = 0.01), CNFA-low (r = 0.8, p < 0.001), CNFrac-SD (r = -0.6, p = 0.04), CNFrac-low (r = 0.6, p = 0.04) and CNFL-low (r = 0.7, p = 0.02). The percentage of MMP-9 correlated with DC density in the entire cohort (r = 0.47, p = 0.03). Conclusions: Corneal nerve measures showed a strong correlation with higher DC density, suggesting that the number of cells maybe be modulated by the corneal nerves in the central cornea. MMP-9 also showed a moderate correlation with DC, supporting an inflammatory role.

Longitudinal changes in Langerhans cell density of the cornea and conjunctiva in contact lens-induced dry eye.

BACKGROUND: The aim was to determine longitudinal changes in Langerhans cell density (LCD) in the human cornea and conjunctiva during asymptomatic and symptomatic contact lens wear. METHODS: Twenty-five participants with contact lens-induced dry eye (CLIDE) and 35 without CLIDE (NO-CLIDE), diagnosed using a range of symptom questionnaires and objective tests (tear film break up, cotton thread tear test and corneal staining) were enrolled. The central cornea and nasal bulbar conjunctiva were examined using a Heidelberg laser scanning confocal microscope at baseline and following one, four and 24 weeks wear of daily disposable hydrogel contact lenses. Twenty-three non-contact lens-wearing controls were also examined. Langerhans cells were counted manually from randomly selected images. RESULTS: In the cornea, mean and standard error of the mean LCD was greater after one week of lens wear in CLIDE (55 ± 7 cells/mm2 ) versus NO-CLIDE (43 ± 4 cells/mm2 ) (p = 0.041) and controls (27 ± 4 cells/mm2 ) (p < 0.001). LCD was also greater in NO-CLIDE versus controls (p = 0.010). At week 4, LCD was greater in CLIDE (41 ± 6 cells/mm2 ) versus controls (27 ± 4 cells/mm2 ) (p = 0.004). There were no other significant differences between groups at weeks four or 24. In the conjunctiva, LCD was greater after one week of lens wear in CLIDE (17 ± 1 cells/mm2 ) (p = 0.003) and NO-CLIDE (17 ± 3 cells/mm2 ) (p = 0.001) versus controls (7 ± 1 cells/mm2 ). There were no significant differences between groups at weeks four or 24. CONCLUSIONS: The initial transient increase in corneal and conjunctival LCD in CLIDE (versus NO-CLIDE) suggests an inflammatory component in the aetiology of this condition.

Characterization of Goblet Cells in a Pterygium Biopsy Using Laser Scanning Confocal Microscopy and Immunohistochemistry.

PURPOSE: To confirm that structures presumed to be GCs observed using laser scanning confocal microscopy (LSCM) are actually GCs. METHODS: A single tissue sample was obtained from a pterygium that was freshly excised from a 33-year-old male. After viewing what were believed to be GCs in the tissue sample using LSCM, the same sample was observed using laboratory confocal microscopy and immunohistochemistry. GCs were identified by a combination of classic morphologic appearance and the use of immunofluorescence to antibodies for mucin 5AC and cytokeratin-7. The LSCM and immunohistochemistry results were compared. RESULTS: Using LSCM, GCs were observed between 7 and 41 µm deep, at the level of the superficial basal cells of the tissue sample. GCs were estimated to have a diameter of 35-40 µm near the surface and 20-30 µm in the deeper layers. A small dark dot was visible in some GCs, indicating cell nuclei and/or the opened apical portion of cells representing the site of mucin release. GCs were more reflective and larger than the surrounding cells. Positively stained GCs in immunofluorescence showed a similar distribution pattern to those observed with LSCM. The tissue sample stained intensely for GC-specific mucin type 5AC. CONCLUSIONS: The pattern of discrete, large reflective cells observed using LSCM are likely to be GCs.

Time Course of Changes in Goblet Cell Density in Symptomatic and Asymptomatic Contact Lens Wearers.

PURPOSE: To investigate longitudinal changes in goblet cell density (GCD) in contact lens (CL) wearers who do and do not develop symptoms of dry eye (DE). METHODS: Sixty healthy individuals fitted with daily disposable hydrogel CLs and 23 age-balanced non-CL-wearing controls underwent assessment using the 5-item dry eye questionnaire, noninvasive tear film break-up time measurement, ocular surface assessment, and phenol red thread test evaluation. Laser scanning confocal microscopy (LSCM) and conjunctival impression cytology (CIC) were used to assess GCD at baseline and follow-up visits at 1 week and 1 and 6 months. After 1 week, all CL wearers were categorized as those who were and were not symptomatic based on responses to the CL dry eye questionnaire-8 (CLDEQ-8). A linear mixed-model was used to examine changes in GCD over time. RESULTS: The global mean GCD of the 83 participants at baseline (before CL wear) was 476 ± 41 and 467 ± 52 cells/mm2 using LSCM and CIC, respectively. After 6 months of CL wear, GCD was reduced by approximately 13% and 29% in asymptomatic (N = 29) and symptomatic (N = 17) CL wearers (all P < 0.001), respectively, observed with both LSCM and CIC. CONCLUSIONS: Contact lens wear induces a reduction of GCD over 6 months, which is exacerbated in those with DE symptoms. Either LSCM or CIC can be used to assess GCD in the conjunctiva.

Assessment of conjunctival goblet cell density using laser scanning confocal microscopy versus impression cytology.

PURPOSE: To determine the association between conjunctival goblet cell density (GCD) assessed using in vivo laser scanning confocal microscopy and conjunctival impression cytology in a healthy population. METHODS: Ninety (90) healthy participants undertook a validated 5-item dry eye questionnaire, non-invasive tear film break-up time measurement, ocular surface fluorescein staining and phenol red thread test. These tests where undertaken to diagnose and exclude participants with dry eye. The nasal bulbar conjunctiva was imaged using laser scanning confocal microscopy (LSCM). Conjunctival impression cytology (CIC) was performed in the same region a few minutes later. Conjunctival goblet cell density was calculated as cells/mm(2). RESULTS: There was a strong positive correlation of conjunctival GCD between LSCM and CIC (ρ=0.66). Conjunctival goblet cell density was 475±41 cells/mm(2) and 466±51 cells/mm(2) measured by LSCM and CIC, respectively. CONCLUSIONS: The strong association between in vivo and in vitro cellular analysis for measuring conjunctival GCD suggests that the more invasive CIC can be replaced by the less invasive LSCM in research and clinical practice.