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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 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 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: 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 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 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 outcome of two different nucleo-cytoplasmic ratio calculations in relation to the number of cells measured from conjunctival impression cytology (CIC) images., Methods: CIC from the exposed nasal bulbar conjunctiva surface was undertaken on 40 young adults using a Millcell(®)-CM filter; the filters were stained with Giemsa, colour images taken of the cells in a monolayer at 200 X magnification, graded by the Nelson scale and then a projection overlay method used to outline the cell and nucleus borders. Nucleo-cytoplasmic ratios were calculated based on literal interpretation of the term (i.e. N/CYT = nucleus area/[cell area - nucleus area]) as well as based on cell area (i.e. N/CELL = nucleus area/cell area)., Results: A range of N/CYT or N/CELL values are expected for all cells, regardless of squamous metaplasia grade, with slightly greater heterogeneity expected from N/CYT calculations. The difference between the two calculations gets progressively less as the squamous metaplasia grade increases. For all grades of cells, these ratios could be determined to within approximately ±1% if adequate numbers of cells were measured (i.e. 75, 65, 50 and 35 for grade 0 to grade 3 cells respectively). If fewer cells are measured, then the variability in N/CELL or N/CYT calculations can be expected to increase., Conclusions: In assessments of nucleo-cytoplasmic ratios, it is important that the calculation used be specified and that the numbers of cells measured be provided.

Purpose: To objectively assess the cell and nucleus dimensions of human bulbar conjunctival cells in female soft contact lens wearers to illustrate a method for assessment of the nucleus-to-cytoplasm ratio based on simple linear measures., Methods: Impression cytology samples were taken from the nasal side exposed bulbar conjunctiva surface from 12 young adult, white European females with a history of successful daily soft contact lens wear. A Millcell(®)-CM filter was used after topical anesthesia, which was stained with Giemsa. Color images of portions of the cells, in a monolayer at 200× magnification by light microscopy, were graded by the Nelson scale and then a projection overlay method was used to outline the cell and nucleus borders. The cell longest dimension (LONG), shorter dimension (SHORT), and the longest dimension of the nucleus (NUCLONG) were measured. A nucleus-to-cytoplasm N:C ratio was calculated from (LONG-NUCLONG)/NUCLONG., Results: Cells had appearances consistent with a grade 2 or 3 squamous metaplasia and were moderately enlarged (mean LONG ± SD of 46.0 ± 3.8 microm), only slightly elongated (mean LONG:SHORT ratio of 1.397 ± 0.101) and the nucleus size was consistently greater than normal (man 12.8 ± 1.3 microm). A calculation of N:C showed a relatively wide range of values with average values from 1:2.143 to 1:3.317 (for an overall mean of 2.675 ± 0.371)., Conclusions: These studies further indicate that grade 2 to 3 squamous metaplasia of the exposed bulbar conjunctival cells is an expected consequence of soft contact lens wear. The cell enlargement is not associated with a significant change in cell shape (i.e., the LONG:SHORT ratio is little different from grade 0 cells) but is associated in a slight increase in nucleus size. The calculated N:C ratio based on linear measures is no higher than 1:5 and more likely closer to 1:2.5.

Purpose: To quantitatively assess the effects of pH on the corneal stroma under an applied hydrostatic pressure., Methods: Sclera-stroma preparations from adult sheep eyes were perfused with isotonic mixed salts solutions at 37 degrees C for 9 hr under an applied hydrostatic pressure and then fixed for electron microscopy., Results: Exposure to pH 7.25 solutions resulted in slight swelling, retention of lamellae, and the keratocytes were intact. The collagen fibril diameter was 40.0+/-3.5 nm, which was the same as non-perfused samples (40.9+/-3.7 nm). Perfusion at pH 5.75 resulted in slight compaction, some disorganization of the lamellae, and almost total destruction of the keratocytes. The collagen fibril profiles in cross section were irregular and reduced to a mean of 30.2+/-3.5 nm (p<0.001)., Conclusions: Exposure of the corneal stroma to slightly acid pH at the so-called critical electrolyte concentration pH for proteoglycan-ionic interactions can cause substantial structural changes.

Purpose: To develop a method for the quantitative analyses of the ring-shaped features usually observed on the surface of mammalian corneal epithelial cells., Methods: The corneas of 10 young adult female albino rabbits were prepared for scanning electron microscopy (SEM) by fixation in 2% glutaraldehyde in 80 mM cacodylate buffer (pH 7.2 to 7.4, 320 to 340 mOsm/kg) and single images taken at 1000x of the central region of the corneal epithelial surface. On enlarged prints at 5000x final magnification, cell-cell borders were marked as were the outlines of the ring-shaped features. Cell areas and the dimensions of the ring-shaped features were measured and the cell characteristics in terms of electron reflex (light, medium, and dark) as well as the number of apices (sides) also noted., Results: The ring-shaped features had an overall density of 2.5/100 microm2, or an average of 6.5/cell. With the corneal epithelial cells being small and light in appearance (median area 36 microm2), medium in size and appearance (median area 151 microm2), or larger and darker (median area 476 microm2), the number of features/cell was very strongly associated with both the cell type (p < 0.001) and the cell area (p < 0.001). The ring-shaped features could be between 0.65 and 5.5 microm in diameter, but sizes more than 3 microm were most unusual. Ring-shaped features of light cells were smallest (median diameter 1.35 microm), slightly larger on medium cells (1.55 microm), and largest on the dark cells (1.90 microm)., Conclusions: The ring-like features seen on SEM photomicrographs of rabbits (also known as craters or holes) are reproducible in terms of their overall density per unit surface area as well as their diameter. Such methods should be useful for objective analysis of these features after various treatments of the ocular surface.

Purpose: To qualitatively and quantitatively assess the size and shape of the surface cells of the bovine corneal epithelium as assessed by scanning electron microscopy., Methods: After quality-control measures on recent postmortem bovine eyes obtained from a slaughterhouse, the ocular surface was aggressively washed with 0.97% saline and then fixed for scanning electron microscopy (SEM) by a dropwise application, in 37 degrees C moist chamber, of a 2% glutaraldehyde solution (in 80 mM cacodylate, pH 7.2 to 7.4). A set of images were taken from the central region of the surface of the corneal epithelium. The cell areas, perimeter and dimensions of 25 to 30 cells from 18 different samples were measured with a digitiser pad or rule, and number of bordering cells (sides) counted manually. Cell shape was assessed by comparing the longer and shorter dimensions (LS ratio) and calculation of the figure coefficient (FC, 4PiA/P2 )., Results: With the washing protocol, surface cells were evident that displayed a range of electron reflexes (light, medium and dark) and that were decorated with microplicae and some crater-like features. These features were generally not evident if the surface was only rinsed with saline. The cell surface areas ranged from 58 to 6444 microm2 (mean 1748 +/- 1169 microm2), and increased as the number of sides increases. Cells with a higher number of sides (n=10) tended to be more rounded (LS ratio averaging 1.24) while cells with small number of sides (n=3) were more elongated (LS ratio 1.94). Shape (FC) values ranged from 0.379 to 0.967, and increased as the number of cell sides increases. Assessment of cell shape in relation to the number of cell sides identified only a small proportion of surface cells with unusually low FC values., Conclusions: With an appropriate preparative procedure (aggressive washing with saline), bovine corneal epithelial surface (squamous) cells can be shown to have a similar appearance by SEM to that previously observed for rabbit and human corneas. The cell sizes are however substantially greater than those seen in rabbits, presumably because of the much larger corneal diameter (c. 28 mm vs. 13 mm). Based on detailed analysis of cell size and shape, the results indicate that the squamous cells have reasonably predictable characteristics.

Purpose: To examine the surface of pre-corneal mucous layer by scanning electron microscopy (SEM) with the view to assessing whether it was an amorphous, fibrous or porous structure., Methods: Healthy female albino rabbits (2 kg) were euthanised at 15.00 h and the corneal surface immediately fixed with buffered glutaraldehyde fixative pH 7.2 to 7.4 at 35 degrees C, carefully rinsed with balanced salts solution (BSS) and then fixed, or treated with the same fixative containing 0.05 to 0.25% w/v cetylpyridinium chloride (CPC). The corneas were critical point dried and gold-palladium coated, prior to examination at 1000x or 15000x magnification., Results: Buffered glutaraldehyde fixation resulted in an SEM image of polygonal cells, with very contrasting electron reflexes (light, medium and dark) and distinct cell surface microplicae and large crater-like features (average diameter 2.8 +/- 0.9 microm). Along the cell-cell borders was a distinct line (referred to as "caulking"), perhaps composed of mucous. Rinsing of the corneal surface with BSS just before fixation reduced the cell contrast, the surface was then covered with very short strands or small clumps of presumed mucus, the cell-cell border features were changed, but the crater-like features were largely unchanged (average diameter 2.5 +/- 0.7 microm). Fixation in the presence of 0.05% or 0.1% CPC resulted in the appearance of very short fine strands and occasional coarse mucous strands or even small plaques of mucous on the cell surfaces. The microplicae and craters were still often evident. Fixation with 0.2 or 0.25% CPC however yielded a surface with a uniform grey reflex that included numerous ultramicroscopic debris particles (average diameter of 72 nm) and was punctuated by numerous pores. No microplicae or craters were evident. The average pore diameter was 70 nm, their density averaged 59/5 microm(2), but a radial distribution analysis indicated no substantial pattern., Conclusions: The pre-corneal mucous layer can be dispersed, by careful rinsing with a divalent cation containing solution, into small fibrils or clumps, but the cell surface craters are unaffected. After precipitation with a low concentrations of a polycation, some coarse fibrils are also formed. The use of high concentrations of a polycation in the fixative transforms the more superficial aspects of the pre-corneal mucous layer into a pore-like gel. The crater-like features therefore do not appear to be mucous, but the pore-like structure may be indicative of the organisation of the mucous layer on the surface of the living eye.

Purpose: To qualitatively and quantitatively assess the surface cells of the bulbar conjunctiva of slaughterhouse-obtained bovine eyes by impression cytology (IC) and scanning electron microscopy (SEM)., Methods: After quality-control measures and careful surface washing of recently enucleated bovine eyes over 20 min, impression cytology samples were taken from the nasal bulbar conjunctiva with a MILLCELL filter, or the ocular surface fixed in a moist chamber by a dropwise application of buffered isotonic glutaraldehyde solution. Impression cytology samples were stained with Geimsa or haematoxylin-periodate-Schiff. Fixed samples, that included the nasal bulbar conjunctiva, were prepared for SEM. From overlays of light microscope images of IC material or SEM images, cell areas, nucleus areas and cell and nucleus dimensions were measured with a digitiser pad., Results: Impression cytology routinely showed large cells with an average area of 1035 +/- 368 microm(2), and a low nucleus-to-cytoplasmic (N/C) ratio of 0.112 +/- 0.048. The cells had dimensions averaging 46 microm (long) and 29 microm (short). Similar areas and dimensions were also found using SEM, which also showed the surface cells to have a range of lighter and darker electron reflexes and to be decorated with microplicae or microvilli. Goblet cells were not evident with either method of viewing., Conclusions: The superficial cells of the bovine bulbar conjunctiva appear to routinely be of a squamous cell phenotype.

Purpose: An adequate volume of tears is considered essential to prevent desiccation of the exposed ocular surface, especially in elderly individuals. Few assessments of tear meniscus height (TMH) have however been reported for elderly individuals., Methods: Close-up images of the lower marginal tear strip were obtained by videography over about 30 s in 56 elderly individuals (38 women, 18 men; 71 +/- 5 years of age) without significant eye disease and having a functional lacrimal system (Schirmer test). From such an "en face" view, the vertical height of the tear meniscus (TMH) at the lower eyelid was measured from stopped video frames. Firstly, TMH was assessed at the mid-point of the eyelid from five frames separated by 5 s; these values were averaged to give a time-averaged assessment of TMH (a tTMH value). Secondly, a position-averaged method (pTMH) was used in which TMH was assessed at 5 locations along the eyelid margin from a single frame that included several mm of marginal tear strip, and an average value calculated. The videographs were also used to assess palpebral aperture and the exposed ocular surface, a 1 min Schirmer test measure (closed eye) was also made, and keratometry data was available for most subjects., Results: A wide range of average values was encountered for both tTMH (range 0.057 to 0.271 mm) and pTMH (0.031 mm to 0.325 mm). The group-averaged tTMH value was 0.172 +/- 0.047 mm (median 0.164 mm) while the group-averaged pTMH value was 0.171 +/- 0.058 mm (median 0.168 mm), with no difference being found between the two methods (p = 0.84, paired t-test). With either method, the apparent TMH value was greater when the exposed ocular surface value (average 1.68 +/- 0.51 cm(2)) was lower and vice versa, and was statistically-significant for the tTMH measures (p = 0.02, r = 0.32), but not the pTMH measures (p = 0.18, r = 0.18). Neither TMH measure was related to the Schirmer test values or to central corneal curvature., Conclusions: The results indicate that healthy elderly individuals can be expected to have a measurable lower marginal tear strip, and that objective assessment of this may be a useful alternative to the Schirmer test. A possible association was found between the measures of TMH and the palpebral aperture height (and thus the exposed ocular surface), and so might need to be considered when TMH is assessed.

Purpose: To quantitatively assess the measured nucleus-to-cytoplasm (N/C) ratios for sets of bulbar conjunctival cells categorised by a subjective grading scheme (0-3)., Methods: Impression cytology samples were taken from the nasal bulbar conjunctiva of 37 individuals (aged 22 to 74 years) without any significant eye disease. The sheets of cells were generally stained with Giemsa, photographed and 35 mm slides prepared. The images were graded 0 to 3 according to the scheme of Nelson [Cornea 7, 71-81 (1988)] and approximately 35 contiguous cells from each image measured by planimetry from an overlay., Results: Analysis of 1097 cells revealed that the N/C ratios ranged from 1.395 to 0.056 and correlated well with the grading. The average N/C ratio (range for +/- 1.96 SD) for each grade was 0.607 (0.983 to 0.531) for grade 0, 0.533 (0.759 to 0. 333) for grade 1, 0.291 (0.427 to 0.155) for grade 2, and 0.154 (0. 228 to 0.080) for grade 3., Conclusions: N/C ratios can be assessed from conjunctival impression cytology samples and appear to be sufficiently consistent to allow for a designated range of cell N/C ratios to be specified for each grade.

The effects of repeated exposures to UV-A (335 nm) and UV-B (305 nm) radiation on the crystalline lens were studied by treating cultured bovine lenses daily or weekly. The effects of irradiation on lens optical quality were monitored using an automated scanning laser system that records both relative transmittance and focal length across the lens. Relatively low radiant exposures of UV-B were used (0.06, 0.03, 0.01 J/cm2) compared to UV-A (1.44 J/cm2). In total, 38 treated lenses and 32 controls were cultured for times ranging from 400-1000 hours. Results indicate that this range of UV-B exposure may represent the threshold for in vitro UV-B induced opacification. Lenses treated weekly with 0.06 J/cm2 UV-B showed a significant decrease in transmittance compared to controls 69 hours after the first treatment and an increase in focal length variability. The ability of the lens to repair itself, as found in a previous single dose study, was absent after repeated doses. Lenses exposed daily to 0.03 and 0.01 J/cm2 UV-B showed no significant change in transmittance or focal length variability compared to controls. Daily exposure to 1.44 J/cm2 UV-A resulted in no significant change in transmittance or focal length variability compared to controls.

Corneas from female pigmented rabbits were prepared for scanning electron microscopy at 15.00 h. The superior-temporal and inferior-nasal quadrants were evaluated at 500X at-stage magnification, normal to the corneal surface, at central, paracentral and peripheral sites. Non-parametric statistical analyses of the histograms of cell surface areas at each site from seven corneas revealed significant differences between the three sites but not between superior and inferior quadrants.

The surface of 8 different corneal quadrants was assessed by scanning electron microscopy. Cells were identified by their electron reflex (light, medium, dark) and their areas, longest and shortest dimensions measured using a manual digitizer tablet/computer-interfaced system. Analyses of over 8200 cells show that while light cells are predominantly small (uncorrected mean area of 39.7 microns2), there can be light cells ten times larger than this. Medium reflex cells show a very wide range of area (uncorrected values ranging from 16 to 1257 microns2) while dark reflex cells show an even wider range (24 to 2218 microns2). Analysis of the longest to shortest dimensions (LS ratio) reveals light cells to be more elongate than medium cells while dark cells tend to be rounder.