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Purpose: To assess variability across 3 measures of central corneal thickness (CCT) obtained with a non-contact specular microscope and taken over a few minutes from habitual soft contact lens wearers., Methods: One eye from 200 healthy adults (with an average age of 21 y, half of whom had a 3.5 ± 2.1 year history of successful daily wear of soft contact lenses while the control group had nominally normal eyes) were assessed using the auto-focus Topcon 2000P instrument to obtain an image of the endothelium and CCT., Results: The individual CCT values encountered in the 200 subjects ranged from 0.449 mm to 0.591 mm, with the average of 3 measures ranging from 0.459 to 0.591 mm in the control group and between 0.449 and 0.585 mm for the SCL wearers. The group mean CCT values were the same for both groups (at 0.524 mm), but the group mean SD value was marginally higher (at 0.028 mm) for the SCL group as compared to controls (SD = 0.026 mm). The normalized intra-subject variability (as the group-mean coefficient of variation, COV value) was 0.843 ± 0.401 for the control group and higher at 1.08 ± 0.546 for the SCL group (p < 0.001)., Conclusions: Repeat measures of central corneal thickness, using a non-contact specular microscope, is very similar to those taken on age-matched non-contact lens wearers. These results may not equally apply to similar pachymetry measures in patients wearing RGP lenses or for older patients wearing soft contact lenses., (Copyright © 2021 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Background: The purpose of this review was to evaluate the consistency of central corneal thickness (CCT) values reported with use of Topcon SP-2000 P and SP-3000 P non-contact specular microscopes since their introduction in 1999 with the two microscopes having been commonly used in a wide range of studies., Methods: As a primary resource, PubMed was used to search for peer-reviewed articles in any language that included CCT values obtained with non-contact specular microscopy reported for humans with nominally healthy corneas. Relevant articles were obtained and any cited publications also checked., Results: A total of 76 articles were identified which reported CCT on different small-to-moderate sized groups of individuals, published between 1999 and 2019. From these, an overall group mean CCT value of 0.525 ± 0.013 mm (median 0.525 mm) can be calculated. An estimated 95 % confidence interval (CI, based on 1.96 SD) would be between 0.500 and 0.550 mm. For the two Topcon models, the group mean ± SD values were 0.529 ± 0.013 mm and 0.517 ± 010 mm respectively. An assessment of the CCT data sets in relation to the reported average age indicated no statistically significant effect (p = 0.289, r = -0.129). Very similar average CCT values were also encountered in 4 other reports where these microscopes were used in large-scale population studies as well as in 2 other reports using the newer Topcon SP-1 P model., Conclusions: The Topcon stand-alone non-contact specular microscopes have yielded consistent and predictable corneal thickness measures over many years., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess the impact of using different numbers of cells in calculations of the coefficient of variation (COV) value for normal and polymegethous endothelia METHODS: Four sets of 20 non-contact specular microscope images obtained from Caucasian individuals were assessed, and categorized according to the extent of polymegethism, i.e. grade 0 (none), grade 1 (mild), grade 2 (moderate) and grade 3 (substantial). Cell areas were measured manually and then values for between 2 and 100 cells were progressively added and averaged to generate COV estimates. These were then assessed in terms of their relative values (as percentages +/- SD) in relation to the value obtained on over 100 cells., Results: For the 4 sets of endothelia with group-mean COV values of 25.8, 33.1, 45.1 and 56.8%, the reliability of the COV estimates realized asymptotic values of close to ±1.0, ±2.7, ±3.6 and ±5.0% with 90 cells, but with greater uncertainty with few numbers of cells, e.g. only to within ±3.4, ±5.3, ±6.1 and ±7.9% with 75 cells., Conclusions: COV estimates for the corneal endothelium are dependent on the number of cells used in the calculations. It is recommended that every effort should be made to not only assess 75-100 cells per endothelial image, but that this number should be the same or very similar for all endothelial images in a particular data set so that the uncertainly (or estimated proportional error) in the estimates is balanced., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Clinical Relevance: Slitlamp-type assessments of eye blink activity with head and chin support need to consider time-related differences that can occur., Background: Previous studies have not assessed the predictability of changes in spontaneous eye blink rate occurring during slitlamp observations., Methods: Video recordings were made of eye blink activity of 85 young adults who were either emmetropic or spectacle wearers for refractive errors between -8.25 D and +8.25 D. After an initial adjustment period of one to two minutes positioned at the slitlamp (including the time after removing spectacles), participants had a five minutes recording made in silence while seated with forehead and chin support and directing their gaze to a high-contrast target on a distant whiteboard under ambient luminance of 35 cd per square metre., Results: The mean spontaneous eye blink rate values over five minutes were 13.4 ± 3.1 blinks/minute (± SD), ranging from 7.4 to 20.8 blinks/minute. Overall, incomplete eye blink events were noted 39 times in the total of 5,704 recorded (that is, 0.68 per cent of all eye blinks). There was a progressive decline in averaged spontaneous eye blink rate values (r = 0.897, p < 0.05), with 70.6 per cent of the participants exhibiting a higher spontaneous eye blink rate value in the first minute compared to the fifth minute. The inter-participant variability in spontaneous eye blink rate also progressively declined over time, but there was no detectable difference in either averaged values or the variability in spontaneous eye blink rate in relation to refractive error., Conclusions: In slitlamp-based assessments of eye blink activity, a small progressive time-related reduction appears likely but is not obviously related to visual blur in ametropic individuals.

Background: Published studies indicate that assessments of goblet cell density using conjunctival impression cytology has provided very variable results, but the reasons for this are unclear. Systematic analyses of the sources of variability are required., Methods: From 20 healthy young adults, conjunctival impression cytology specimens were obtained using a supported filter unit applied to the superior bulbar conjunctiva. The filters were stained with Giemsa and 10 non-overlapping, randomly selected high-power field images were obtained from each specimen and the numbers of goblet cells per high-power field counted., Results: From all 200 high-power fields assessed, the numbers of goblet cells ranged from zero to 74, with an overall mean value of 11.6 ± 14.8 per high-power field. From each successive set of 10 microscope field images from all individuals, the average number of goblet cells ranged from 23.2 in the first high-power field that obviously included numerous goblet cells down to 6.2 per high-power field. As the outcome from multiple counts/individual was systematically increased, these averages progressively decreased from 23.2 to 11.6 per high-power field, and while the standard deviation values also progressively declined (from 7.9 to 5.5 per high-power field), the relative variability (as the co-efficient of variation) did not, and increased to averaged values of over 100 per cent., Conclusions: These analyses indicate that there is a benefit of making multiple counts of goblet cells from different high-power fields, but that there is no obvious benefit of using more than five to seven high-power fields for any particular specimen., (© 2019 Optometry Australia.)

Purpose: To assess goblet cell size and numbers in relation to the extent of multilayering of conjunctival impression cytology (CIC) samples as a basis for reducing variability in image selection for goblet cell density (GCD) estimates., Methods: CIC was undertaken immediately postmortem off the superior bulbar conjunctiva of healthy young adult rabbits onto Millicell-CM Biopore filter units. After fixation with buffered glutaraldehyde and Giemsa staining, two × 200 images were selected from each sample representative of either slight multilayering or substantial multilayering, projected at × 1000, an overlay of the outlines of the goblet cells was made, and their dimensions and areas were measured., Results: From measures of 4918 goblet cells, the average value (+/- SD) for the longest dimension was 17.7 ± 6.4 μm and 14.6 ± 5.3 μm for the shortest dimension. The GCD values ranged from 210 to 2069/mm 2 , with a mean of 1074 ± 601/mm 2 , but was lower for slightly multilayered images (at 537 ± 239 cells/mm ) compared with multilayered regions (at 1612 ± 601 cells/mm 2 ; p < 0.001). The measured areas ranged from 72 to 491 μm 2 , with average values from any particular image ranging from 110 to 370 μm 2 , which were inversely correlated with the estimated GCD (Spearman's rho = - 0.722, p < 0.05)., Conclusions: Larger goblet cells but in fewer numbers were predictably found across the filter surface where there were fewer layers of cells and vice versa. This difference could be considered in selection of images for counts of goblet cells from CIC specimens.

Background: In vivo confocal microscopy (IVCM) has been used for over 10 years to assess the goblet cell density (GCD) within the human conjunctiva, but the reported values have been variable with no obvious indications as to why., Methods: From publications between 2008 and 2019, representative GCD values were extracted, as well as on the image sampling strategy used., Results: Average GCD values for any particular group of individuals ranged from 7 to 979 goblet cells / sq. mm, and with one notable outlier removed, an overall group-mean value for GCD (+/- SD) from single site locations was 207 +/- 143 goblet cells / sq. mm from 15 data sets for those usually designated as control subjects, with a value of 190 +/- 161 goblet cells / sq. mm calculated from 20 single site data sets from other (patient) groups. An overall analysis indicated that the reported average values for GCD from different groups of individuals increased according to the number of images assessed / individual (Spearman rho = 0.304), on the number of individuals evaluated to generate an averaged value for each group (rho = 0.367), and the total number of images assessed (rho = 0.346, multivariate analysis partial r = greater or = to 0.522)., Conclusions: In the use of confocal microscopy to assess the number of goblet cells in the human bulbar conjunctiva, the substantial differences reported appear to be linked to the protocols used for image selection, and some type of standardization needs to be developed., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

This study was to assess the impact on the cornea and eye blink activity of adapting rabbits to continuous lighting (CL) compared to a 14:10 light:dark cycle. Female New Zealand White rabbits (2 to 2.5 kg) were maintained under a light: dark (L:D) cycle or switched to continuous fluorescent lighting (CL) for an average of 17 +/- 2 days. Animal behaviour in their cages was manually recorded using an event marker and in vivo slitlamp biomicroscopy at 40× undertaken in mid-afternoon. Animals were then euthanized and the corneas prepared for scanning electron microscopy (SEM). From images taken at 500× from the central region of the corneas, the number of exfoliating (desquamating) cells and the relative number of different cells with light, medium or dark reflexes were assessed for the corneal epithelial surface, while the number of cells/unit area were assessed for both corneal epithelium and endothelium. Exposure to continuous lighting was associated with higher number of eye blink events (15.7 vs 8.2/15 min) and mild corneal surface alterations evident by biomicroscopy with higher numbers of intra-epithelial 'granules' (32 +/- 14 vs. 4 +/- 3/sq. mm). SEM revealed low numbers of exfoliating cells on the corneal epithelial surface in all CL-adapted animals, but not in L:D controls. Trends were observed for there to be slightly higher numbers of epithelial cells/unit area, higher numbers of small light reflex cells and lower numbers of larger dark reflex cells in CL animals. The corneal endothelium showed no obvious adverse effects in CL-adapted animals but the percentage of 'hexagonal' cells was slightly higher compared to L:D controls. The results indicate that even a short period of exposure of laboratory-raised rabbits to constant lighting can be associated with mild adverse effects on the corneal epithelial surface., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Clinical instruments using Scheimpflug image-based methods to obtain optical sectional images of the cornea have been introduced in recent years along with proposals that it should be possible to routinely and reliably measure the optical density (referred to as the densitometry) of the human cornea in situ. Such a concept is reviewed from the perspective of what might be considered as the basic principles underlying the understanding of corneal transparency (from the 1950's) and the progressive changes in these ideas from subjective slitlamp-based clinical observations from the late 1960's, especially in contact lens wearers. Much more has been learned about the overall macrostructure (including corneal thickness) and the ultrastructure of the cornea from contemporary studies in the 1990's, and these aspects of the cornea will be reviewed alongside consideration of the methods of assessing the optical characteristics of the cornea in the living eye. From these perspectives, in this review systematic consideration will be given to what objective (quantitative) output one of these Scheimpflug-based systems provides and how this information might be actually related to corneal transparency characteristics that might be observed clinically, particularly after long-term contact lens wear., (Copyright © 2018 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To evaluate whether visual target character and visibility affects spontaneous eye blink rate (SEBR) in primary eye gaze and silence., Methods: Video recordings were made of young healthy adults who were either emmetropic (n=32) or who wore spectacles for refractive error (range -4.75D and +4.50D (n=31). Emmetropes had 5min recordings made whilst seated and looking towards a distant whiteboard. For spectacle wearers, recordings were made whilst looking towards the whiteboard with a 35mm sized cross, and repeated after spectacle removal. The average number of eye blinks over 5min was assessed, and its intra-subject variability as the coefficient of variation (COV)., Results: Over 5min without a distance target, an average SEBR of 10.4blinks/min was observed in emmetropes with a of COV=38.1%, and a significant increase in SEBR over the 5th minute to 13.6blinks/min. Hyperopes being asked to look towards a distant target showed the essentially same blinking rate of 11.1/min with or without spectacle wear with the intra-subject variability (COV) being 21.3%. Myopic subjects showed a slightly higher SEBR if looking towards a target without their spectacles (12.4 vs. 11.0blinks/min), with the COV being 18.8%., Conclusions: The studies indicate that some form of visual target could be useful to promote constancy of spontaneous eye blink activity over time, but that a distance visual target (when provided) does not need to be seen clearly., (Crown Copyright © 2018. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: To assess agreement between 3 measures of central corneal thickness (CCT) taken over a few minutes from nominally normal eyes with a non-contact specular microscope., Methods: 100 eyes from 100 healthy adults (with an average age of 22 y) were assessed using the Topcon 3000 P instrument to obtain a high quality image of the endothelium and pachymetry., Results: The group mean CCT values from the 1st, 2nd and 3rd measures were 0.519, 0.520 and 0.520 mm, but the sets of values could differ by between - 0.020 mm and + 0.029 mm, i.e. between - 4.0 and + 5.6% of the average values. Paired comparisons (e.g. 2nd vs. 1st set) indicated limits of agreement (LoA) to be between - 0.020 and + 0.020 mm of the averaged value. Across the 3 measures, the averaged SD was 0.005 mm to give an estimate of the intra-subject variability (as the coefficient of variation, COV) of 0.91% (range 0 to 2.3%). The variability in the pachymetry measures was not predictably related to the averaged values of CCT (r = 0.022) or self-reported refractive error of the subjects (r = 0.012). Some repeat pachymetry assessments obviously included the same region of the endothelium., Conclusions: Single pachymetry measures with a non-contact specular microscope are only likely to be able to generate CCT estimates within +/- 4% of the expected average values. This repeatability is comparable to single image estimates of endothelial cell density and therefore acceptable in most cases., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

The goal of this review was to identify and discuss the specialized methods that have been used to assess the corneas of the eyes of living rabbits exposed to the damaging effects of ultraviolet-B (UV-B). From publications reviewed between 1916 and 2018, both albino and pigmented rabbits were used, usually being young adults weighing between 2 and 2.5 kg, and with many recent studies carried out under sedation. Older assessments were generally based on the use of in vivo slitlamp examinations, sometimes with the use of fluorescein or rose bengal to identify damaged cells, supported by light microscopy (histology) of excised corneas. In later years (after 1960), these structural studies have included in vivo and ex vivo specular microscopy, transmission and scanning electron microscopy as well as impression cytology. Early studies included measurements of the thickness of excised corneal specimens but in vivo pachymetry methods were widely used from the 1980s. Other assessments have included measurement of light transmission spectra of excised corneas, measures of metabolites in isolated corneas or staining corneal specimens for enzyme activities. While a wide range of specialized methods have been used, most of them have provided only descriptions of the effects of UV-B., (© 2018 The American Society of Photobiology.)

Purpose: To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry., Methods: Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A step-wise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations., Results: Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/image in markedly polymegethous endothelia only increased the variability in the calculations., Conclusions: These analyses indicate that substantial region variability in cell area values can be expected in polymegethous endothelia. The analysis further confirm that using only small numbers of cells (e.g. less than 50/image) in such cases is likely to yield far less reliable estimates of COV.

Several older studies on rabbits indicated that the pre-corneal tear film was unusually stable and this perspective was revisited recently. However, the methods used for these studies were very different from those generally used in human studies. A literature search was undertaken for the time period of 1965 through 2017, mainly using PubMed, to identify studies where values for the tear break up time (TBUT) were reported for the eyes of nominally normal (healthy) laboratory rabbits regardless of breed or age and where the methods were more similar to those routinely used in human clinical studies. For 20 reports identified where sodium fluorescein was used, the average TBUT values in any particular study ranged from 1.9 to 51 s, with a group mean of 21.8 ± 11.9 s (SD), with the inter-study variability in TBUT (as the coefficient of variation) being 19.4%. For four studies not using fluorescein, the mean break up time reported was 32.7 ± 16.2 s, while a separate study (also not using fluorescein) reported an average break up time of 1788 s. Most reports of the pre-corneal tear film stability in laboratory rabbits, especially as reported over the last 10 years, indicate break up times of less than 60 s have been observed, although has been little consistency in the methods used. Overall, this outcome is not consistent with a perspective that the rabbit (as routinely used in experimental studies) has an extraordinarily stable tear film.

Purpose: To assess the impact of using different microscope magnifications for the goblet cell density (GCD) estimates from conjunctival impression cytology (CIC) samples from healthy individuals METHODS: In a prospective study, CIC specimens were collected from the superior bulbar conjunctiva (12 o'clock, 5mm from limbus) of 20 adult subjects (average age 22 years) onto Millicell-CM membranes and Giemsa stained. A region from each CIC filter containing reasonably high numbers of goblet cells was imaged by light microscopy at a final magnification of 400X and then the same region assessed at 200X and then 100X. The images were enlarged, the goblet cells marked and counted and GCD values/sq mm calculated., Results: The mean GCD estimates at 400X magnification, 200X and 100X were 644±180, 405±72 and 365±81 cells/sq mm respectively, and these values were statistically different (p<0.001)., Conclusions: As a result of non-uniform distribution, a strategy to select a 400X high power microscope field (HPF) that appears to include a moderate number of goblet cells will have a probability (by at least 20:1) that the GCD estimates will likely be higher compared to those at 200X or 100X, and the probability for higher GCD values is at least 15:1 comparing assessments made at 200X to 100X. Investigators should use only one magnification, with that of a medium power field (200X final magnification) likely being the most useful., (Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Background: To evaluate oculo-mandibular interactions during evaluation of spontaneous eye blink rate (SEBR) of normal young adult human subjects while seated at a slitlamp., Methods: Repeat video recordings of five minutes duration were made on 76 young adult emmetropic subjects aged 18-25 years. The subjects were instructed to direct their gaze horizontally toward a distant target with the entire cornea of the left eye illuminated with a broad beam cobalt blue light. Repeat recordings were made: (a) on the following day in silence in group one; (b) immediately in silence for group two; and (c) immediately either while holding their mouth open slightly or while responding to casual conversation for groups three and four., Results: For group one the averaged SEBR values for the first and second recordings were 13.5 and 14.6 blinks/minute, and similar results were obtained for group two if the subjects were relaxed (for example 13.9 and 12.0 blinks/minute). A slight, but statistically significant, time-related decline in SEBR was usually noted. For non-relaxed (restless) subjects exhibiting spontaneous mouth and jaw movements while being videographed, the averaged SEBR values were around 27 blinks/minute. For groups three and four, analyses of those individuals who managed to maintain a mouth open posture for the video recordings, the averaged SEBR was 7.2 blinks/minute, while those engaged in casual conversation had an averaged SEBR of 20.3 blinks/minute., Conclusions: Mouth and jaw movements and a non-relaxed state can substantially affect spontaneous eye blinking., (© 2017 Optometry Australia.)

Purpose: To develop and validate an easily procured objective estimate of the cell density of the superficial epithelial cells of the normal bulbar conjunctiva using conjunctival impression cytology (CIC)., Methods: From 20 healthy non-contact lens-wearing young adults, CIC was undertaken off the nasal aspect of the exposed bulbar conjunctiva, the filters stained with Giemsa, and images taken at 200× that only included an all-but-contiguous monolayer of epithelial cells. These images were projected at 1000× magnification and an overlay drawn of the location of the stained nuclei, from which two types of analysis were undertaken using either 100 micron circular or square regions of interest (ROI). From the former, the distances between all nuclei were measured and the number of nuclei/circle calculated to establish uniformity of cell nuclei distribution as a necessary prerequisite to using a grid-based counting method. From the latter, the number of nuclei in each square grid was manually counted and the grid-by-grid variability assessed., Results: The average inter-nucleus distance was 16.1+/3.5 microns, with predictable positioning to successive nuclei in the circular ROI to give an estimated numerical density of 1944 ± 237/sq mm. Counting of nuclei in grid squares yielded numbers between 12 and 42 to yield an estimated cell density of 2390 +/ ̶330 /sq mm, with repeat counts off grid square ROIs being predictably within +/-15%., Conclusions: These studies indicate that it should be fairly easy to use carefully selected CIC samples to make estimates of the density of the superficial conjunctival cells. Such a method should be useful and offer advantage over the subjective assignment of morphological grades to CIC samples which have been shown to be highly variable.

Purpose: To assess variability in endothelial cell density (ECD) estimates when polymegethism (variance in cell areas) is present., Methods: Using noncontact specular microscope images of the corneal endothelium, 4 sets of 20 cases were selected, which included 200 cells and had coefficient of variation values of less than 30% (group 1), 31%-40% (group 2), 41%-50% (group 3), and over 50% (group 4). A stepwise analysis was undertaken, 20 cells at a time, of the ECD estimates when using different numbers of cells for the calculations., Results: The net differences in ECD estimates when comparing sets of 20 cells with 200 cells were 5.0% ± 3.9%, 8.1% ± 7.3%, 11.3% ± 9.4%, and 14.5% ± 12.4% for groups 1 to 4, respectively. For measures on 100 cells per image, the predicted variances in ECD values were 5.6%, 8.8%, 11.1%, and 13.7% for the 4 groups., Conclusions: Higher values of corneal endothelial polymegethism result in predictable increases in the variability (uncertainty) in ECD estimates, thus reducing the "accuracy" of ECD values. There is no obvious utility in assessing more than 100 cells in such endothelia.

Objective: To assess the morphological details of the acini of the normal meibomian gland., Animals Studied: Six young adult pigmented rabbits., Methods: The upper eyelid was prepared in extended configuration by glutaraldehyde fixation. Tissue block sections approximately 0.5-1 mm from the eyelid margin were assessed by light microscopy in sagittal sections and transmission electron microscopy (TEM) in coronal sections. TEM images at between 1000× and 2000× magnification were enlarged onto A3-sized prints and cell size and nuclei measured by planimetry., Results: Light microscopy sagittal sections revealed clusters of variable sized acini, sometimes appearing to be slightly overlapping and without any obvious spatial organization of the internal cells (meibocytes). The estimated areas of the acini were close to 6500 sq micron. Coronal sections, as examined by TEM, allowed for visualization of small to large acini (average diameter 82 ± 17 microns, with an estimated area of 5500 sq. microns) containing variable numbers of immature (partly differentiated) or mature (fully differentiated) meibocytes with a distinct spatial organization. The average area of the meibocytes was 158 ± 81 square microns, and they usually appeared to have a single nucleus (with an average sectional area of 29 ± 12 square microns). Within individual acini, peripherally located immature meibocytes tended to be smaller and have higher nucleo-cytoplasmic area ratios, while more centrally mature located meibocytes tended to be slightly larger and had lower or much lower nucleocytoplasmic ratios., Conclusions: Comparative studies on meibomian glands can be undertaken with objective assessments to assess for normality or abnormality., (© 2016 American College of Veterinary Ophthalmologists.)

Purpose: The aim was to assess differences in eyeball mass, corneal diameter and central corneal thickness in slaughterhouse-procured ovine eyes., Methods: Over a 12-year period, measurements of eye globe mass, horizontal corneal diameter and central corneal thickness were routinely undertaken within two hours post-mortem. Only eyes free of obvious mechanical damage or disease were used., Results: From measurements on 736 quality-selected and trimmed eyes, globe wet mass ranged from 10.4 to 25.2 g, horizontal corneal diameter from 19.0 to 26.5 mm and central corneal thickness measured by ultrasonic pachymetry from 0.543 to 0.836 mm (with an overall average of 690 ± 0.056 mm). The ocular globe mass was strongly correlated to horizontal corneal diameter (r 2  = 0.829). Central corneal thickness correlated with globe mass (r = 0.543) and to horizontal corneal diameter (r = 0.402). Based on the different anatomical measurements, a lamb's eye would be expected to have a thinner cornea (average 0.640 mm) than that of an adult outbred ewe (average 0.730 mm). In freshly procured eyes showing signs of slight corneal oedema, central corneal thickness was greater (average 0.856 ± 0.052 mm) and up to 24 hours of cold storage resulted in predictable increases in central corneal thickness of six to 24 per cent, especially in eyes showing signs of corneal oedema before storage., Conclusions: Based on the correlations obtained, differences in ovine eyes can be attributed to growth-related differences in the animals and thus, indirectly to their expected ages. A simple measure of the horizontal corneal diameter in ovine eyes used for laboratory studies would be a useful indicator in reporting these studies., (© 2016 Optometry Australia.)

Purpose: To assess if polymegethism and pleomorphism were evident in corneal endothelium after medium-term rigid gas permeable (RGP) contact lens wear., Methods: In a cross-sectional observational study over 12 years, single images of the central region of the corneal endothelium of one eye of 46 subjects were taken with a non-contact specular microscope, along with a measure of central corneal thickness (CCT). The images were printed onto A3-sized paper and 100 cells/image measured by planimetry., Results: Subjects aged between 20 and 32 years, with an average cumulative RGP wear of 6.0+/- 1.6 years (range 3-9 years) were assessed; 26 of the subjects were Caucasian and 20 were Asian. The mean CCT was 0.515+/- 0.027mm. The group cell area value was 401+/- 42 sq micron to give an estimated endothelial cell density (ECD) of 2520+/- 273 cells/sq mm. As compared to a historical database, most endothelia (37/46) showed some changes with the mean coefficient of variation on cell area (COV) being 36.7+/- 8.0% and the percentage of 6-sided (HEX) being 51.8+/- 8.8%. There were modest correlations between years of RGP wear and both COV (p=0.009, r spearman=0.424) and HEX (p=0.025, r spearman=-0.291), but not for ECD or CCT., Conclusions: Corneal endothelial polymegethism appears to be a commonplace consequence of RGP lens wear with the magnitude of the change being related to the cumulative duration of the lens wear., (Copyright © 2016. Published by Elsevier Ltd.)

Purpose: To investigate the possible association between body stature (height) and corneal thickness and radius in younger-adult Caucasians, especially within the context of previously published literature., Methods: Body height and weight were measured in 109 healthy subjects, with an average age of 24 ± 6 years (mean ± SD). Subjects underwent an ophthalmic assessment including anterior segment imaging by Scheimpflug topography and specular microscopy. Central and peripheral corneal thickness and corneal radius were analyzed. The relationship between body stature and corneal parameters was assessed using simple and multiple regression analysis. Effect size was determined by generating regression and correlation coefficients., Results: Body height ranged from 1.54 to 1.86 m (mean ± SD 1.67 ± 0.08 m), central corneal thickness from 465 to 629 μm (554 ± 33 μm), whereas corneal radius measured between 7.16 and 8.49 mm (7.75 ± 0.24 mm). Body height was weakly associated with central corneal thickness and peripheral corneal thickness (r ≥ -0.180), and moderately with corneal radius (r = 0.351). Based on the regression equations, central corneal thickness decreases by 8 μm, whereas corneal radius increases by 0.11 mm for each 0.1-m difference in body height. No significant correlations were found for similar assessments using body weight or body mass index., Conclusions: Differences in corneal radius and corneal thickness can be linked to body stature. However, effect sizes were consistently small and no more than 13% of the variability in corneal curvature could be explained by variations in body stature.

Purpose: To assess whether or not the numbers of goblet cells were different across regions of a conjunctival impression cytology (CIC) specimen., Methods: CIC specimens were obtained from the exposed nasal bulbar conjunctiva from 22 healthy young Caucasian adults, stained with Giemsa and evaluated at a final magnification of 200× (medium power fields). Up to 14 different non-overlapping microscope fields, depending on whether or not a field was without goblet cells, were randomly selected., Results: A microscope field could contain between 0 and 145 goblet cells. If only fields that included goblet cells were analyzed, the mean goblet cell counts (22 specimens, 10 fields/specimen) ranged from 37.8 to 44.8/field (equivalent to 259-313 goblet cells/mm 2 ). When 3, 5 or 10 fields were used to calculate average goblet cell counts for each specimen, the standard deviation values were between 15.8/field and 21.7/field, with the lower values obtained if 10 fields were used. The resultant averaged inter-sample variability, as the coefficient of variation (COV), ranged from 40.0% to 57.5%, while the averaged intra-sample variability in counts ranged from 52.1% to 73.9%. If fields without goblet cells were also used, the resultant mean goblet cell count (from 10 fields/specimen) was statistically lower at 33.1/field (232 goblet cells/mm 2 )., Conclusions: These analyses confirm various subjective comments made by early CIC investigators; the distribution of goblet cells across a CIC filter may be highly variable.

Purpose: The aim was to compare efficacy of treatments for diabetic macular oedema (DMO) from changes in visual acuity (VA) and central macular thickness (CMT)., Methods: Peer-reviewed articles from 2004 to 2014 reporting intravitreal injections of bevacizumab (IVB), ranibizumab (IVR) or triamcinolone acetonide (IVTA) or laser photocoagulation therapy (LPT) provided data on pre-treatment (baseline) and final outcome measures. Net changes and relative changes (percentage) were assessed by linear regression analyses., Results: From 88 data sets the overall net change of VA was -0.10 ± 0.12 logMAR (mean ± standard deviation), being -0.13 ± 0.11 logMAR for IVB, 0 ± 0.08 logMAR for IVR and -0.12 ± 0.08 logMAR for IVTA as compared to 0.01 ± 0.14 logMAR for LPT. For CMT, the overall net change was -103 ± 71 microns, being -108 ± 64 microns for IVB, -182 ± 73 microns for IVR, and -102 ± 57 microns for IVTA and was -49 ± 60 microns for LPT. Overall, modest correlations were found between the absolute central retinal (macular) thickness change and the VA change, and the relative changes in these measures (p < 0.001, r = 0.522 or 0.457). The predicted visual outcome from a 100 microns reduction in CMT was -0.083 logMAR units, an effect not substantially influenced by the CMT measurement method., Conclusions: Pharmacological treatment of DMO can be expected to result in a predictable decrease in CMT with an accompanying increase in VA, with the overall outcome being better than laser treatment., (© 2016 Optometry Australia.)

Background: The aim was to assess the variability of spontaneous blink rate (SBR) with and without a chin and forehead support., Methods: Forty-eight healthy non-contact lens wearers, aged from 20 to 39 years, had five-minute video recordings made under ambient lighting of 350 to 400 lux, while directing their gaze to a distant target at head height. Half the subjects (group 1) were seated resting against the chair head rest and the other half (group 2) seated but with chin and forehead at a slitlamp. The first 35 blinks were analysed in detail., Results: As assessed over five minutes, 35 to 111 blinks were counted, with SBR between 6.9 and 21.8 blinks per min (average 13.9 per min). Over the initial 35 blinks, the average momentary SBR values (calculated from the inter-blink intervals) averaged 24.8 blinks per minute in group 1 and 19.3 blinks per minute in group 2 (not significantly different, p = 0.273) but a statistically significant (p < 0.001) decrease in SBR was observed over the initial 10 to 15 blinks in group 2. The variability in momentary SBR values, as assessed from successive blinks, had coefficient of variation (COV) values of 80 and 78 per cent, respectively over 35 blinks., Conclusions: Averaged spontaneous blink rates over short time periods (that is, five minutes) should be suitable to compare various experimental paradigms but if very short periods are used (for example one minute or less), then there could be significant time-related changes, especially when a subject is seated with chin and forehead support., (© 2016 Optometry Australia.)

Purpose: To review the special pharmacology of tetracycline antibiotics as anti-inflammatory drugs for treatment of obstructive Meibomian gland disease (MGD) METHODS: PubMed was used as principal resource for articles, regardless of language, on doxycycline and minocycline with key interests being on their serum and tissue pharmacokinetics and their use in clinical studies as part of management of MGD., Results: With oral dosing of between 50 and 200mg, peak blood levels of these antibiotics have been reported to be predictably dose-dependent at between 1 and 5 microgram/mL, with human tear film levels not being detectable with 100mg dosing of doxycycline but levels of 0.2 microgram/mL with 200mg minocycline. That these two tetracycline antibiotics reach the conjunctiva is indicated by conjunctival pigmentary changes due to photosensitization after very long term use. Based the reported use in a range of clinical studies on MGD, dosing with these two antibiotics for MGD is likely to be useful at relatively low doses (e.g. 100mg for doxycycline or 50mg for minocycline, either at once or twice daily depending on severity at presentation and previous history) continued for 2 to 3 months, with the expected outcome being small-to-substantial decreases in abnormal appearance of the glands (from -4 to -89%) and increases in tear film stability (from 21 to 273%)., Conclusions: Oral doxycycline and minocycline have predictable pharmacokinetics and have been reported to improve Meibomian gland dysfunction over a few months of use., (Copyright © 2015 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To objectively compare sizes of corneal epithelial surface cells visualized by scanning electron microscopy (SEM) or collected by impression cytology (IC)., Methods: Corneas of recent postmortem sheep eyes were either glutaraldehyde fixed for SEM or IC samples taken, glutaraldehyde fixed, and then stained with Giemsa. The corneal epithelial surface was examined at ×200, with the borders of all cells marked and dimensions and areas measured. For SEM, the cells were classified as having light, medium, or dark electron reflex and by the number of cell sides., Results: SEM revealed a mosaic of polygonal features with different electron reflexes according to the density of cell surface microplicae. The cell area and longest dimension were predictably dependent on the number of cell sides. Compared with the overall average cell area of 1598 μm(2) (range 200-3900 μm(2)), the dark reflex cells were predominantly the largest, with an average area of 2435 μm(2) and having the average longest dimension of 59.9 μm(2). Cells collected by IC had a range of areas from 1150 to 4800 μm(2) but were predominantly large and had an average area of 2802 μm(2) with the average cell longest dimension averaging 64.4 μm(2). IC-sampled cells had nucleocytoplasmic ratio values consistent with their having a squamous phenotype., Conclusions: Cells sampled by IC from the corneal epithelial surface are predominantly those that are enlarged (and flattened out) in preparation for desquamation, a feature also attributed to the dark reflex cells seen in SEM.

Purpose: To assess the repeatability of corneal thickness (CT) measurements, along the horizontal meridian up to 5mm from centre, in healthy eyes with the Pentacam instrument., Methods: CT was measured in 82 right eyes (82 healthy subjects) at 11 corneal locations nominally 1mm apart along the horizontal meridian with Scheimpflug topography Pentacam. Two consecutive scans were performed in quick succession. The repeatability of CT was determined by assessing differences between measurements and calculating the coefficient of variation (CV). The relative repeatability (difference) was calculated as the ratio of the absolute peripheral CT difference to the central thickness. A variant of Bland-Altman analysis was carried out to determine the effect of the overall magnitude of CT on the absolute and relative differences., Results: Mean CT (μm) at the temporal 5mm location was 770 ± 51 and 823 ± 56 μm at the corresponding nasal location; central CT was 554 ± 36 μm. Good repeatability (CV < 1.2%) was found at all corneal locations. Differences (arithmetic; absolute and relative) were 3 ± 17 μm; 13 ± 11 μm; 2 ± 2%; respectively temporally and -1 ± 15 μm; 13 ± 8 μm; 2 ± 1% nasally, both at 5mm from the corneal apex. These values decreased for central CT (0 ± 6 μm; 4 ± 4 μm; 1 ± 1%). Differences between both measurements in all corneal locations were not statistically significant (p = 0.308 ANOVA Games-Howell)., Conclusions: Pentacam shows good repeatability for pachymetry measurements up to 5 mm away from the corneal apex across the horizontal meridian, even though repeatability decreases slightly towards the periphery. A single Pentacam scan will be sufficient for most clinical applications., (Copyright © 2015 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Nucleus-to-cytoplasm (N:C) characteristics of published images of human bulbar conjunctival cells designated as showing severe (grade 3) squamous metaplasia were retrospectively assessed. From publications over a 40-year period, measurements were made from images of cell and nucleus size (based on areas and dimensions) and four different calculations were made for nucleo-cytoplasmic (N:C) ratios. From 54 published images, the mean nucleus-to-cytoplasm area ratio (NU/CYT AREA ratio) was 0.145 +/- 0.077 (range 0.052 to 0.346), compared to two different reference set values of 0.069 +/- 0.017 and 0.080 +/- 0.021. Similarly, a nucleus-to-cytoplasm length ratio (as LNLONG) was 0.308 +/- 0.080 (range 0.191 to 0.475) compared to reference values of 0.226 +/- 0.032 and 0.236 +/- 0.034. Similar differences in reference values were obtained using two other N:C ratio calculations. A wide range of values was found for morphometric N:C indices from published images, especially those without a scale bar, indicating a high incidence of inconsistent grading assignments. Overall, only about 30% of the published images showed morphological features consistent with severe squamous metaplasia (i.e., with the rest neither enlarged nor showing substantially changed N:C ratios) with no substantial pictorial evidence indicating that cells from the human bulbar conjunctiva have pyknotic nuclei. Current evidence indicates that grade 3 squamous metaplasia cells should be substantially enlarged., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The purpose of this study was to assess the apparent goblet cell density (GCD) from conjunctival impression cytology (CIC) samples in relation to the number of conjunctival cells collected onto the filters. CIC specimens were collected from the superior-temporal bulbar conjunctiva of 16 pigmented rabbits onto Biopore (Millicell-CM) membranes, fixed with buffered glutaraldehyde and stained with Giemsa. Different numbers of microscope fields of view in each of the specimens were imaged by light microscopy using a 20× magnification objective lens (200× final magnification), and the goblet cells marked and counted. The GCD values/sq. mm were calculated. The same conjunctival region of 3 other rabbits was also prepared for transmission electron microscopy (TEM) by fixation, in situ, with the same buffered glutaraldehyde. Mean values for GCD estimates were found to vary from 399 to 1576 cells/sq. mm, depending on the image sampling and analysis strategy chosen, with the lowest inter-sample variance of around 10% being found if a maximum goblet cell count was taken on substantially multilayered regions of the CIC specimens. Counts of the number of goblet cells per 1000 visible conjunctival epithelial cells yielded a value of close to 90 (range 36-151), with modest inter-sample variability of around 30%. A three or ten 200× microscope field and random sampling strategy yielded mean GCD values between 542 and 670 cells/sq. mm, but with very high intra- and inter-sample variance of at least 60% and sometimes higher than 100%. TEM confirmed the multilayered organization of the conjunctiva and the deeper lying goblet cells. The general use of a goblet cell count as an objective marker for conjunctival normality or health is likely to be highly variable unless a more specific strategy is adopted. Beyond providing details of exactly the counting strategy used, it would be very useful to provide full details of the actual microscope field size used as well as information on the intra-sample variability in goblet cell counts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: The aim was to objectively assess size, nucleus and nucleo-cytoplasmic ratio features of squamous cells from the corneal epithelium, Methods: The corneas of recent post-mortem sheep eyes were either glutaraldehyde-fixed for transmission electron microscopy or impression cytology samples taken, glutaraldehyde-fixed and stained with Giemsa. From the specimens for impression cytology, a representative region was photographed from 12 different samples taken from the central region and 16 different samples taken from mid-peripheral regions of the corneal epithelium. Images were subjected to morphometry after overlays were generated., Results: Electron microscopy revealed a very distinctive stratified corneal epithelium with several superficial layers, confirming the squamous phenotype. Impression cytology from such superficial layers revealed a cell size of 60.1 ± 4.8 μm, nucleus dimension of 12.3 ± 1.5 μm, cell area of 2,419 ± 416 μm(2) and nucleus area of 131 ± 31 μm(2) . A nucleo-cytoplasmic ratio based on nucleus-to-cell length had a mean of 0.207 ± 0.022, while a cytoplasm-to-nucleus length ratio was 3.975 ± 0.474. Estimates of the nucleo-cytoplasmic ratio based on areas had a mean value of 0.059 ± 0.011. Very similar results were found for mid-peripheral corneal epithelium., Conclusions: The results strongly indicate that the squamous phenotype of the superficial corneal epithelial cells is characterised by a large size, large nucleus and low nucleo-cytoplasmic ratio. These morphological characteristics show a notable resemblance to data obtained from impression cytological studies on human conjunctival epithelial cells showing severe squamous metaplasia., (© 2015 The Author. Clinical and Experimental Optometry © 2015 Optometry Australia.)

Purpose: To investigate the asymmetry of the peripheral cornea up to 5 mm nasally and temporally from the centre and to assess correlations with regional peripheral corneal thickness., Methods: Central and peripheral corneal thickness was measured by Scheimpflug imaging (Pentacam) in 113 eyes of 113 healthy, pre-presbyopic Caucasian subjects. Absolute and relative corneal thickness were analysed in 1 mm steps up to 5 mm to the nasal and temporal sides with the corneal apex as the central reference point. Nasal-temporal asymmetry was calculated as the thickness ratio between corresponding off-centre thickness measurements., Results: The mean (±SD) central corneal thickness was 552 ± 36 μm. CT increased by 22% at 4 mm temporally to 672 ± 44 μm, and 32% at 4 mm nasally to 731 ± 45 μm. The nasal-temporal asymmetry became greater with increasing distance from the corneal centre, with a mean difference of 59 ± 22 μm at 4 mm from the apex. The nasal-temporal thickness ratio, based on this difference, was significantly related to the relative temporal (r = -0.41, p < 0.001, simple linear regression) and nasal corneal thickness (r = 0.61, p < 0.001)., Conclusions: A substantial and progressively increasing nasal-temporal asymmetry in corneal thickness has been confirmed by Scheimpflug imaging, which is related to the magnitude of corneal thickness at peripheral locations. Pachymetry output data and models, including volume calculations, that assume symmetry to the corneal thickness profile may not provide optimum metrics for planning and predicting the outcome of corneal refractive surgery procedures., (© 2014 The Authors Ophthalmic & Physiological Optics © 2014 The College of Optometrists.)

Purpose: To assess fluorescence emission properties of sodium fluorescein-rose bengal mixtures in buffered aqueous solution., Methods: Solutions of sodium fluorescein (NaFl) or rose bengal (RB) or mixtures of these two chemicals were prepared over a range of dilutions in 1% NaCl with 10mM phosphate buffer to give a pH of 7.5 at room temperature. Absorbance and fluorescence spectra were recorded in 10mm path length cuvettes., Results: The fluorescence emission from NaFl extends between 480 and nearly 600nm, a spectral range that is also covered by the absorbance of RB (between 500 and 580nm). With very dilute solutions of NaFl (less than 0.002%), an apparent total quenching of its fluorescence can occur in the presence of 0.01% RB, with a proportionate decrease at lower concentrations of RB., Conclusions: The presence of RB in an aqueous solution of NaFl at physiological pH appears to act in a similar way to a barrier filter, resulting in the quenching of the measurable fluorescence from NaFl. It remains to be established how substantial or significant such an effect might be if a mixture of NaFl and RB as used as part of the examination of the external eye., (Copyright © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess the morphology of superficial bulbar conjunctival cells collected by impression cytology or by scraping., Methods: Freshly procured sheep eyes from a slaughterhouse were washed and then conjunctival impression cytology (CIC) samples taken from the nasal aspect of the bulbar conjunctiva on a Biopore Millicell filter unit, or cells scraped from the same location and smeared onto glass slides. After alcohol fixation, all samples were stained with Giemsa. Cell images were obtained by light microscopy at 200× magnification, and subjected to morphometry., Results: Cells obtained by scrape/smear processing appeared larger and less densely staining than cells procured by CIC. This appearance was confirmed by morphometry with measures of cell longest dimension LONG being 27.7 ± 1.3 μm and 21.7 ± 1.5 μm, respectively, while cell AREAC measures averaged 543 ± 42 μm(2) and 333 ± 51 μm(2); these differences were significant (p < 0.001). Similarly, cell nucleus length (NUCLONG) and nucleus area (AREANUC) were also larger in smeared cells (p < 0.001). Various calculations of nucleo-cytoplasmic ratios as based on dimensions or area were only slightly different when comparing cells obtained by the two methods, indicating that the glass slide-smeared cells simply spread out more than cells on the CIC filters., Conclusions: Differences in cell and nucleus size can result from use of different processing methods. Therefore, in making comparisons between sets of conjunctival cells, it is important that cell collection and processing methods be considered.

Purpose: To objectively assess the morphology of superficial conjunctival surface cells showing squamous metaplasia as seen in contact lens wearers., Methods: Impression cytology samples were taken from interpalpebral bulbar conjunctiva from 23 young adult white European women with an average of 6 years of successful daily soft contact lens wear and 23 non-contact-lens-wearing women of the same age. Cell images were graded, and cell and nuclear dimensions and areas were measured by planimetry., Results: The contact lens wearers had a mean squamous metaplasia grade (Nelson) of 2.74 versus 0.35 for the non-lens wearers, with the cell longest dimension (LONG) being 54.4 ± 5.6 μm versus 22.4 ± 2.9 μm, respectively (P < 0.001). Similar differences were found for the cell shortest dimension (SHORT) and cell area values (P < 0.001). The cell nuclei in the contact lens wearers, based on measures of cell nuclear length (NUCLONG), were 12.7 ± 1.7 μm versus 10.0 ± 1.1 μm (P < 0.001) with no evidence of pyknotic (shrunken) nuclei. The nucleo-cytoplasmic ratios had mean values of 3.603 versus 1.282 for the cytoplasm-to-nucleus length (CYT/NUC length) ratio, and 0.075 and 0.319 for the NU/CYT length ratio for contact lens wearers and non-wearers, respectively., Conclusions: As squamous metaplasia develops, at least in long-term soft contact lens wearers, the conjunctival surface cells become considerably enlarged, but the nuclear size of these cells also increases, rather than showing signs of pyknosis.

The purpose of the study was to assess spatial separation of goblet cell orifices observed at the surface of the rabbit bulbar conjunctiva by scanning electron microscopy (SEM) specimens of the bulbar conjunctiva from 8 healthy pigmented rabbits were obtained using a special preparation technique by which the tissue was carefully stretched out during glutaraldehyde fixation. On high magnification prints of SEM images of the conjunctival surface, the locations of goblet cell openings (orifices) to the apical surface were marked and the centre-to-centre spacing between all such orifices measured. Across the regions of interest (ROI), with averaged dimensions of 322 μm × 230 μm (adjusted for tissue shrinkage), the averaged value for the distances between all orifices was 196 μm (range 141-241 μm), with the calculated density of orifices being 412 mm(-2). A sequential order-based analysis of the spatial separation between orifices indicated a predictable value of 6 μm, a separation that showed a nearly linear inter-dependence over distances of at least 200 μm. The openings of goblet cells to the surface of unstimulated bulbar conjunctiva have a organized spatial distribution that is consistent with there being an organized control of goblet cell secretion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To further evaluate the spontaneous eyeblink rate (SEBR) of healthy adult human subjects according to direction of gaze, especially in the presence of bright light reflective glare., Methods: On 32 subjects aged between 18 and 24 years, separate video recordings of 5 min duration were made with different conditions of gaze (horizontal, slightly upwards or slightly downwards) under normal lighting or a distant lighting glare source., Results: The SEBR in primary eye gaze under normal lighting was 11.7 +/- 0.9 eyeblinks/min with a coefficient of variation (COV) of 20.5 %. A non-significant decrease in SEBR was noted with downward gaze, but a slight significant increase with upward gaze in both SEBR (to 13.0 +/- 1.1 eyeblinks/min) and COV (average 26.1 %). In the presence of glare, SEBR in primary eye gaze increased significantly (p < 0.001) to 14.4 +/- 1.3 eyeblinks/min, with an obvious time-related progressive increase (p < 0.001). On upward gaze in the presence of a glare stimulus, SEBR progressively increased even further (average 15.0 +/- 2.4 eyeblinks/min; p < 0.001), as did the COV (to 29.2 %)., Conclusions: The results indicate that spontaneous eyeblink activity in silence can be affected by the presence of a glare light source, especially if the subjects are looking slightly upwards. This scenario should be avoided, if at all possible, in assessments of spontaneous eyeblink activity.

Purpose: The aim was to assess the morphology of the Asian corneal endothelium, particularly the index of polymegethism and its relationship to endothelial cell density assessments., Methods: The central regions of the corneal endothelium of 72 healthy, normal weight, non-contact lens wearers were assessed by non-contact specular microscopy. There were six groups of persons, aged between 18 and 27 years, whose ethnic origins were self-reported as Asian (Mainland China, Hong Kong, India, Malaysia, Pakistan or other Asian groups). Single images including at least 100 contiguous cells were obtained from each subject and were processed by planimetry after manual border marking to minimise errors. Calculations were made of the coefficient of variation of cell area (COV; polymegethism) as well as the endothelial cell density and percentage of six-sided cells., Results: The COV values ranged from 18 to 30.4 per cent (group mean 24.5 ± 2.8 per cent). The corneas had essentially normal thickness (0.530 ± 0.026 mm), with the endothelia showing a high percentage of six-sided cells (66.3 ± 7.1 per cent) and none showing even mild signs of polymegethism. Endothelial cell density estimates based on average cell areas ranged from 2,020 to 3,623 cells per mm(2) and from 2,157 to 3,835 with calculations based on individual cell areas. The difference between the two estimates of endothelial cell density was strongly related to the coefficient of variation (r = 0.765)., Conclusions: This result is consistent with several older reports indicating that it is uncommon to find polymegethism in healthy young adult Asian individuals. Nonetheless, even low coefficient of variation values can substantially affect the estimations of the cell density., (© 2014 The Author. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.)

Purpose: To review outcomes of studies where fluorescein-tear breakup time (fTBUT) measurements had been made before and after treatment with artificial tears., Methods: Peer-reviewed articles were identified that reported average values for fTBUT before and after approximately 1 month of treatment of dry eye with aqueous-based artificial tears. 48 data sets were used for calculating the net (percentage) change in fTBUT, with 21 including information of variability (as reported SD of average value) of the fTBUT data suitable for a meta-analysis., Results: Prior to treatment, average fTBUT values ranged from 0.9 s to 11.8 s (group mean 4.7 +/- 1.7 s, n = 48 data sets). After treatment, these values ranged from 2.2 to 10.7 s (group mean 6.1 +/- 2.3 s; P<.001), with net increase for the treatment effect being 1.4 +/- 1.2 s (range - 1.4 to + 4.5 s). Similar changes were reported regardless of the type of product type. The meta-analysis confirmed a net effect at 1.3 s, with the magnitude of the variability (as a weighted SE) having a similar overall value of 1.1 s., Conclusions: Following use of 'artificial tears' by dry eye patients, a small but predictable increase in tear film stability has generally been reported, the net magnitude of which is close to a 33% improvement., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To assess how reproducible collagen fibril spacing might be in the corneal stroma as viewed by transmission electron microscopy by calculating averaged values for the 2-D organization., Methods: One cornea from 8 albino rabbits (2 kg) was fixed in situ to preserve natural shape. Thin sections were stained with 2% phosphotungstic acid (PTA) and images taken of fibrils from the central-posterior stroma. After projection at 250,000 X magnification, an overlay was prepared of the fibrils. Using a 500 × 500 nm region of interest (ROI), the distances to all fibril centers were measured to 2 nm resolution., Results: The sets of fibrils had average diameters between 32.4 and 36.1 nm (group mean ± SD of 34.4 ± 4.2 nm). The mean fibril density was 396 ± 21 per square micrometer, with a fibril area fraction of 38.7 ± 3.9%. The mean distance to the literal nearest neighbor fibril center was 43.2 ± 4.5 nm. A radial distribution analysis showed a distinct nearest neighbors peak at 51 nm. This nearest neighbors peak had an average amplitude of 2.236 ± 0.315, with a broader secondary peak being evident in all data sets centered at 93 nm with an average amplitude of 1.166 ± 0.093 (or 53.3 ± 7.1% of the nearest neighbors peak)., Conclusions: Overall, these results show that a predictable 2-D organization can be demonstrated for collagen fibrils in rabbit corneas when consideration is given to sample selection and preparation and the image analysis strategy.

Purpose: To review the use of ophthalmic rose bengal to assess the ocular surface, especially considering the grading scales used., Methods: A literature search was undertaken to identify reports where either rose bengal or lissamine green had been used as a 'vital' stain, with a special interest in identifying studies that provided data before and after treatment for dry eye and also considered the mechanism of action of these two chemicals., Results: Between 1985 and 2006, numerous clinical studies used a consistent grading scheme (that attributed to Van Bijsterveld) to assess the outcome of artificial tear treatments on dry eye patients. With such consistency, including the adoption of a treatment period of 1 month, comparisons can be made to indicate the efficacy of rose bengal staining to assess reduction in ocular surface desiccation. However, in the following years, several alternative grading schemes have been used for both rose bengal and lissamine green and assessment periods have been variable so making inter-study comparisons considerably more difficult to undertake. An attribute of rose bengal appears to be its ability to stain the nuclei of cells, but whether this also occurs for lissamine green is unclear., Conclusions: Ophthalmic rose bengal has been successfully adopted for use to assess the ocular surface over many years as a vital stain. More research is needed to assess whether lissamine green ocular surface staining can simply be substituted for rose bengal in evaluation of dry eye treatments., (Copyright © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess the corneal endothelium, particularly the polymegethism feature, using the Topcon SP-3000P specular microscope with newer center-dot software., Methods: Forty-eight healthy, normal weight, noncontact lens wearers of Asian ethnicity were assessed. Single endothelial images from each subject were processed with center-dot software, reevaluated after correction of obvious errors, and then by manual border marking and planimetry. Endothelial cell density based on average cell area and the coefficient of variation (COV) of cell area (polymegethism) were calculated., Results: Error sources are associated with erroneous location of cell borders (usually creating larger or smaller "cells") or failure to assign cell borders to a marked cell. On the initial application of the center-dot software, the endothelial cell density values ranged from 1822 to 3244 cells per square millimeter (mean, 2644 cells/mm); this range was reduced (eg, 1955-3054 cells/mm; mean 2690 cells/mm on editing or in manual planimetry). The COV values ranged from 17% to 39% (mean, 27.5% ± 5.5%), with one third of the endothelia yielding COV values of greater than or equal to 30%. On editing or in manual planimetry, the COV values were reduced to between 17% and 29% (mean, 24.5% ± 3.2%; P < 0.001)., Conclusions: In the use of a center-dot endothelial analysis program with cell border identification, it is likely that at least 1 set of editing steps is required to produce reasonable results.

Introduction: The Surveillance Epidemiology and End Results Program 2000-2006 data reported that African American women experienced a higher incidence of early-onset invasive breast cancer and in situ breast cancer in comparison with other racial/ethnic groups. Of the total of 25,100 cases reported; in situ breast cancer represented 6,460 cases and invasive breast cancer represented 18,640 cases. This intervention focused on women younger than 40 years to increase understanding and the utilization of preventive services., Method: Over a course of 10 months, the researcher designed, implemented, and evaluated an evidence-based breast cancer course intervention integrating the health belief model. The methodology was implemented in 4-week cohorts through in-class and asynchronous learning-centered environments tailored to African American women aged 20 to 39 years at 2-year and 4-year postsecondary schools and culturally relevant community-based organizations., Results: The intervention was statistically significant, improving understanding of breast cancer in African American women in a classroom environment, p < .039, and in an online environment, p < .05. The study compared ages 20 to 29 and 30 to 39, finding high agreement and no statistically significance difference related to age about the use of preventive services, importance of preventive services, and tailoring preventive service for younger women., Conclusions: The intervention demonstrated the implementation of an evidence-based breast cancer intervention that improved understanding of breast cancer in this race/ethnicity. Additionally, there is a strong need to improve preventive services through the integration of evidence-based educational interventions targeting women of African American prior to age 40.

Background: The aim was to assess the corneal endothelium, post-operative visual outcome and complications following keratoplasty for keratoconus., Methods: Seventy-six corneal grafts (57 per cent penetrating keratoplasties and 43 per cent deep anterior lamellar keratoplasties) were assessed as part of routine follow-up at the Ophthalmology Department at Gartnavel General Hospital in Glasgow. Routine ophthalmic assessment included visual acuity, slitlamp biomicroscopy and scanning slit confocal microscopy of the central cornea. Case records were reviewed and demographic, surgical and post-operative data retrieved. Linear and logistic regression models were used to determine associations between endothelial cell density, visual acuity and post-operative complications. Kaplan-Meier analysis was used to estimate the probability for grafts to retain more than 700 endothelial cells per mm(2) and to have 0.3 logMAR or better vision. Odds ratios were calculated to assess the risk for low cell counts and poor visual outcome., Results: The mean and standard deviation of the endothelial cell count was predictably higher in lamellar (2033 ± 643 cells per mm(2)) than penetrating keratoplasties (898 ± 380 cells per mm(2)) and the mean post-operative cell count for grafts without post-operative complications was higher (1585 ± 775) than those with complications (1312 ± 747 cells per mm(2)). Mean visual acuity was 0.25 ± 0.20 and 0.29 ± 0.41 for lamellar and penetrating keratoplasties, respectively. A higher number of post-operative events was associated with an increased risk for graft rejection (odds ratio 2.40, p = 0.008, multiple logistic regression) and with poorer visual outcome (odds ratio 1.38, p = 0.044). After keratoplasty, the visual outcome and endothelial cell density were not associated, either in penetrating or deep anterior lamellar grafts (p > 0.05)., Conclusions: In keratoplasties performed for keratoconus, visual outcome cannot be predicted reliably by post-operative endothelial cell density but post-operative events can lead to poorer visual outcomes in both types of grafts., (© 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.)

Purpose: To further evaluate possible effects of lighting conditions on the spontaneous eyeblink rate (SEBR) of normal young adult human subjects., Methods: A baseline 5 min video recording was made followed by a second recording under test conditions in 7 different groups of 10 subjects. These test conditions were a simple repeat recording under reference conditions (subjects maintaining silence, with gaze directed towards a target on a 2 m distant whiteboard with the luminance of the reflected light of 35 cd/m, recording under lower (5 cd/m), higher (75 cd/m) and floodlit (150 cd/m) levels, after pupil dilation with phenylephrine 2.5 %, after sudden increase with floodlights (to 200 cd/m), as compared subjects engaged in conversation., Results: The overall SEBR values ranged from 8.4 to 15.8 eyeblinks per minute (mean 12.3 ± 2.8 eyeblinks per minute). These did not significantly change with repeated assessments, no significant changes were seen comparing lower or higher illumination levels or after pupil dilation, but an increase to 20.0 ± 4.6 eyeblinks per minute was seen with a sudden increase in illumination. Conversation increased SEBR to 22.2 ± 6.4 eyeblinks per minute., Conclusions: Spontaneous eyeblink activity in silence may be slightly affected by background lighting conditions, but such effects are notably less than SEBR changes that can occur when there is a sudden increase in lighting levels (as a glare stimulus) or when assessments are made during conversation.

Purpose: To assess density and spatial distribution of the goblet cell orifices at the surface of the rabbit bulbar conjunctiva as an indicator of functional activity., Methods: Specimens of the superior or inferior bulbar conjunctiva from six healthy young adult (2 kg) pigmented rabbits were obtained using a special preparation technique by which the conjunctiva was carefully stretched out during fixation with buffered glutaraldehyde. The apical surface of the specimens was examined by scanning electron microscopy. From high magnification prints, the areas and dimensions of 32-49 orifices/image were measured. In addition, the centre-to-centre spacing and spatial distribution of the orifices were assessed using a nearest neighbors principle., Results: The bulbar conjunctival surface is composed of polygonal cells decorated with surface microplicae and in between which are individual goblet cell orifices. The goblet cell orifices are characterized by tending to be oval in shape (long:short dimensions ratio of 1.57 +/- 0.23) and usually having a distinct line of microvilli around the perimeter. The orifices had a wide range of areas (from 13 to 188 μm(2); group mean +/- SD of 54 +/- 36 μm(2)), and distribution of orifice areas was skewed or even bimodal. The overall orifice density was 387 +/- 68/mm(2), with the group-averaged nearest neighbors distance being 34 +/- 3 μm. Comparisons of the measured nearest neighbors distances to that for an optimum spacing based on numerical density reveals the goblet cell orifices to be slightly further apart and that they were not obviously in groups or clustered., Conclusions: Goblet cell orifices at the bulbar conjunctival surface, a presumed indicator of functional secretory activity, appear to have reproducible density and a discrete and reasonably predictable spatial distribution.

Purpose: To assess the staining of Chang conjunctival epithelial cells with rose bengal and lissamine green., Methods: A human (Chang) conjunctival epithelial cell line was grown on plastic plates in 10% foetal calf serum-supplemented medium in 5% carbon dioxide-air at 37°C. Cells were examined daily between days 2 and 10 of culture, reaching confluence after 7 days, for overall appearance before and after exposure for 3 min at 37°C to solutions of either rose bengal or lissamine green (dissolved in saline, PBS or balanced salts solution). Measurements of nucleus and cell size were made on some confluent cultures., Results: Chang cells, regardless of the growth state or the vehicle used, stain very intensely with rose bengal, with the nucleus of the cells showing the most notable dye uptake. Cultures showing such intensive staining with rose bengal were consistently non-staining with lissamine green. Nucleus size, based on measurements of the long dimension (NUCLONG) were the same for rose bengal or lissamine green exposed cells (averaging 19.5±2.4 μm and 18.8±2.9 μm, respectively). Cell size at confluence, based on measurements of the long dimension of lissamine green-exposed cells (LONG), averaged 40.5±7.2 μm. The cytoplasm-to-nucleus ratio (based on LONG-NUCLONG/NUCLONG) averaged 1.177±0.406 (i.e. approximately a 1:1 ratio)., Conclusions: Cultured Chang conjunctival cells stain intensely with rose bengal, but the same is not seen with lissamine green. The cell morphology is indicative of flattened epithelial cells., (Copyright © 2012 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)