Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats.

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats' corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter-CF-spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.

Evaluation of Tear Evaporation Rate in Subjects with a High Body Mass Index.

AIM: This study evaluated the tear evaporation rate (TER) in subjects with a high body mass index (BMI) and tested the correlation between BMI and dry eye. METHODS: Thirty male subjects aged 18 - 38 years (26.4 ± 4.0 years) with a high BMI (26.4 - 47.0 kg/m2) were enrolled in the study. In addition, a control group of 30 males aged 20 - 36 years (24.0 ± 3.6 years) with a normal BMI (< 25 kg/m2) participated in the study for comparison. Each subject completed the ocular surface disease index (OSDI), followed by the TER measurement using a handheld VapoMeter. RESULTS: Significant differences (Wilcoxon test; p = 0.002 and 0.001) were found between the median scores of the OSDI (10.3 [3.7] vs. 5.9 [7.2]) and TER (35.5 [13.1] vs. 15.5 [12.8] g/m2 h) in the study and control groups, respectively. The OSDI indicated dry eye in 60.0% of subjects in the study group (n = 18). The TER measurements showed that 76.7% of subjects in the study group (n = 23) had dry eyes. A medium correlation (r = 0.569; p = 0.001) was found between the OSDI scores and TER measurements. CONCLUSIONS: The means of the tear evaporation rate and the ocular surface disease index scores are significantly higher in subjects with a high BMI compared with the control group. Therefore, a high BMI is considered a risk factor for dry eye. The measurement of the tear evaporation rate using a handheld VapoMeter is a simple and rapid method to detect dry eyes in combination with other tools.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment.

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.

Assessment of Tear Film Parameters in Smokers and Subjects with a High Body Mass Index.

SIGNIFICANCE: The current study compares the ocular tear film parameters in three different groups using a single noninvasive, practical, and easy-to-use tool. PURPOSE: This study aimed to assess the tear film in smokers, those with a high body mass index (BMI), and healthy subjects using the EASYTEAR view+. METHODS: Thirty men with a high BMI (>25 kg/m2; 24.4 ± 6.4 years), 30 smokers (25.1 ± 6.1 years), and 30 healthy subjects (22.2 ± 3.5 years) were recruited. Each subject completed the Ocular Surface Disease Index, followed by the assessment of noninvasive tear breakup time, tear meniscus height (TMH), and lipid layer patterns (LLPs). RESULTS: Significant differences were found in the median TMH scores between smokers and healthy subjects (P = .03) and between subjects with a high BMI and the healthy ones (P = .04). The median LLP score was significantly (P < .001) higher in normal subjects (4.0 [1.0]) than in smokers (2.4 [1.0]) and subjects with high BMI (2.0 [1.3]). For subjects with a high BMI, the noninvasive tear breakup time score was strongly correlated (Spearman rank correlation coefficient; r) with TMH (r = 0.552, P = .002) and LLP (r = 0.555, P = .001). The LLP showed that grade B (lipid layer thickness, 30 to 50 nm; more compact) was common in subjects with a high BMI, grade C (50 to 80 nm, gray waves) was predominant in smokers, and grade D (~80 nm, dense white-blue layer) represented the majority of normal eye subjects. CONCLUSIONS: Smokers and individuals with a high BMI showed significantly lower lipid layer grades and tear meniscus height scores compared with the control group. The assessment of tear film parameters using the EASYTEAR view+ supports the findings of previous studies that implicate smoking and high BMI as risk factors for dry eye.

The Effect of the Addition of Electrolyte Solutions on the Ferning Patterns of Tears Collected from Normal Eye Subjects.

SIGNIFICANCE: The current study is the first report to describe the improvement of ferning patterns of human tears using electrolyte solutions. The results can help in the production of new artificial tears to improve the quality of tears. PURPOSE: This study aimed to investigate the effect of the addition of different volumes of various electrolyte solutions on ferning patterns of human tears. METHODS: Tear samples (20 µL) were collected from the right eye of 13 subjects (5 men and 5 women) aged 19 to 36 years (27.1 ± 5.1 years) with normal eyes. Then, 1 µL of each tear sample was dried on a microscopic glass slide, and obtained ferns were observed using light microscopy and graded using the 5-point tear ferning (TF) grading scale. Homogenous mixtures of each tear sample (0.5 µL) and different volumes (0.5 to 5 µL) of each electrolyte were prepared. A sample (1 µL) of each mixture was dried, and the ferns obtained were graded and compared with those of the corresponding tears collected from subjects before the addition of electrolyte solutions. RESULTS: After the addition of electrolyte solutions, the TF grades of tears collected from healthy humans were generally improved. Significant (Wilcoxon test) improvements have been seen in the TF grades of the tear samples after the addition of a solution of potassium chloride (P = .03), calcium chloride (P = .01), magnesium chloride hexahydrate (P = .002), and sodium dihydrogen phosphate (P = .002). No significant improvements in the TF grades were seen after the addition of sodium chloride solution (P = .33). CONCLUSIONS: Ferning grades of human tears improved with most of the electrolytes used.

Investigation of Ocular Tear Ferning in Controlled and Uncontrolled Diabetic Subjects.

OBJECTIVES: To investigate the ocular tear film in controlled and uncontrolled diabetes mellitus (DM) subjects and the relationship between dry eye and diabetes. METHODS: One hundred twenty-four diabetes adults (65 male and 59 female, with 62 controlled and 62 uncontrolled DM) were assessed by McMonnies dry eye questionnaire, tear break-up time (TBUT), phenol red thread (PRT) test. In addition, tear samples were collected from the lower tear meniscus of right eyes and dried onto glass slides under controlled room temperature (25°C) and humidity (40%). The glass slides were observed using a digital microscopy and the tear ferning (TF) patterns observed were graded based on Masmali TF grading scale. Patients with a HbA1c value below 6.5% were defined as "controlled DM" and those with a HbA1c value above 6.5% were defined as "uncontrolled DM." The Spearman's correlation coefficient was used to determine the relationship between various tests. RESULTS: Several subjects having dry eye varied depending on the test used. McMonnies questionnaire scores suggested that 28 subjects (23%) showed dry eye (score >14.5), of whom 23 subjects (82%) showed uncontrolled diabetes. The TF patterns showed that 44 subjects (35.5%) showed dry eyes (Grade ≥2), of whom 32 subjects (72.7%) showed uncontrolled diabetes. Phenol red thread test found that 46 subjects (37%) showed dry eye (wetting <10 mm), of whom 38 subjects (82.6%) showed uncontrolled diabetes. Tear break-up time found that 57 subjects (46%) showed dry eye (time <10 sec), of whom 45 subjects (78.9%) showed uncontrolled diabetes. In controlled diabetic subjects, TF grades showed medium negative correlations with both TBUT and PRT (-0.374 and -0.349, respectively). In uncontrolled subjects, TF grades have a strong negative correlation with TBUT (-0.539) and a medium negative correlation with PRT (-0.410). CONCLUSIONS: Dry eye disease is common in diabetic subjects, particularly in people with poor diabetic control. Uncontrolled blood glucose level seemed to be a risk factor for dry eye syndrome. Tear ferning test showed a correlation between diabetes and dry eye in diabetic subjects and can be used as a reliable test to evaluate the quality of the ocular tear film.

Ultrastructure Features and Three-Dimensional Transmission Electron Tomography of Dhub Lizard (Uromastyx Aegyptia) Cornea and Its Adaptation to a Desert Environment.

We report ultrastructural features and transmission electron tomography of the dhub lizard (Uromastyx aegyptia) cornea and its adaptation to hot and dry environments. Six corneas of dhub lizards were fixed in 2.5% glutaraldehyde and processed for electron microscopy and tomography. The ultrathin sections were observed with a JEOL 1400 transmission electron microscope. The cornea of the dhub lizard is very thin (~28-30 µm). The epithelium constitutes ~14% of the cornea, whereas the stroma constitutes 80% of the cornea. The middle stromal lamellae are significantly thicker than anterior and posterior stromal lamellae. Collagen fibril (CF) diameters in the anterior stroma are variable in size (25-75 nm). Proteoglycans (PGs) are very large in the middle and posterior stroma, whereas they are small in the anterior stroma. Three-dimensional electron tomography was carried out to understand the structure and arrangement of the PG and CFs. The presence of large PGs in the posterior and middle stroma might help the animal retain a large amount of water to protect it from dryness. The dhub corneal structure is equipped to adapt to the dry and hot desert environment.

Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission Electron Tomography.

We report the ultrastructure and 3D transmission electron tomography of collagen fibrils (CFs), proteoglycans (PGs), and microfibrils within the CF of corneas of patients with macular corneal dystrophy (MCD). Three normal corneas and three MCD corneas from three Saudi patients (aged 25, 31, and 49 years, respectively) were used for this study. The corneas were processed for light and electron microscopy studies. 3D images were composed from a set of 120 ultrastructural images using the program "Composer" and visualized using the program "Visuliser Kai". 3D image analysis of MCD cornea showed a clear organization of PGs around the CF at very high magnification and degeneration of the microfibrils within the CF. Within the MCD cornea, the PG area in the anterior stroma was significantly larger than in the middle and posterior stroma. The PG area in the MCD cornea was significantly larger compared with the PG area in the normal cornea. The CF diameter and inter-fibrillar spacing of the MCD cornea were significantly smaller compared with those of the normal cornea. Ultrastructural 3D imaging showed that the production of unsulfated keratin sulfate (KS) may lead to the degeneration of micro-CFs within the CFs. The effect of the unsulfated KS was higher in the anterior stroma compared with the posterior stroma.

Repeatability and Diurnal Variation of Tear Ferning Test.

OBJECTIVES: To investigate tear ferning (TF) test repeatability between sessions by observing changes in the tear fern pattern during the day. METHODS: Twenty-three healthy young adults (15 men and 8 women), ranging in age from 20 to 32 years (mean ± SD: 22.9 ± 3.3 years) without signs or symptoms of dry eye disease, ocular disease, or contact lens wear were enrolled in the study. Schirmer I, tear break-up time (TBUT) test, and McMonnies questionnaire were used to screen volunteers. Schirmer I and TBUT tests were applied to both eyes in each subject. Four samples of tear fluid were collected from the right eye of each subject using glass capillaries at set intervals during a single day (9 AM, 11 AM, 2 PM, and 4 PM). The TF patterns obtained from samples were classified according to the Masmali TF grading scale to increments of 0.1. RESULTS: The median values obtained from the McMonnies, Schirmer, and TBUT tests were 4.0 ± 2.0, 30.0 ± 7.0 mm (OD), and 16.0 ± 10.0 sec (OD), respectively. There were no statistically significant differences between the TF grades for tear samples collected at different times of the day (Wilks' Lambda, P = 0.351). The majority (84.8%) of TF grades was between 0.0 and 1.5; the remaining 15.2% of subjects had TF between grades 1.6 and 1.9. The overall mean grade for the TF was 1.1 ± 0.3. There were small insignificant correlations between TF grades and the McMonnies questionnaire (r = 0.1.30) and TBUT (r = 0.248) and a negligible correlation with Schirmer test (r = -0.046). CONCLUSIONS: The results found no significant differences within the TF for tear samples collected at different times of the day, suggesting that there is little diurnal variation evident.

Changes in ultraviolet transmittance of hydrogel and silicone-hydrogel contact lenses induced by wear.

OBJECTIVES: To evaluate the ultraviolet (UV) transmittance spectra of different contact lenses and assess the effect of wear on UV transmittance values and ocular protection factor (PF). METHODS: Transmittance in the UV range (200-400 nm) of 216 contact lenses was measured before and after wear, using a spectrophotometer. The contact lenses used were ACUVUE TruEye, ACUVUE MOIST, ACUVUE OASYS, ACUVUE 2, ACUVUE DEFINE, DAILIES AquaComfort Plus, AIR OPTIX for astigmatism, FreshLook ONE-DAY lens, and Neo Cosmo. RESULTS: This study indicates that the following contact lenses transmitted less than the American National Standards Institute (ANSI) of 5% or less of UVB: ACUVUE DEFINE, ACUVUE MOIST, ACUVUE 2, ACUVUE TruEye, and ACUVUE OASYS. All lenses also met the ANSI standard of less than 30% transmittance of UVA except for AquaComfort Plus, AIR OPTIX for astigmatism, and Neo Cosmo. FreshLook meets the UVA transmission standard but fell short of the UVB transmission standard by 3%. FreshLook and ACUVUE MOIST had the highest calculated PF (3.9) of the hydrogel group and were unaffected after wear. Of the silicone-hydrogels (SiHs), the PF for AIR OPTIX for astigmatism was the highest (10.6) before wear and increased after wear to a PF of 13.3. The results also show that, for the UVC region, significant differences exist in transmittance values before and after wear of all SiH lenses and all hydrogel lenses, except for Neo Cosmo. FreshLook and Neo Cosmo were the only lenses that showed no statistically significant differences in the UVA transmittance after wear. CONCLUSIONS: Ultraviolet-blocking contact lenses were effective in blocking ultraviolet radiation to safe levels recommended by ANSI, whereas non-UV blockers such as AquaComfort Plus and AIR OPTIX for astigmatism also showed UV-attenuation capabilities greater than the values previously reported. The Neo Cosmo contact lens transmitted greater than 86% UVB and 89% UVA, making it unsuitable for UV protection seekers. Transmission is modified after contact lens wear in almost all lenses but more so in the SiH lenses, probably because of the formation of biofilms on the lens surface.

Investigation of tear osmolarity using the TearLab Osmolarity System in normal adults in Saudi Arabia.

OBJECTIVES: To investigate the repeatability and average tear osmolarity using the new lab-on-chip TearLab technology device in normal subjects living in Saudi Arabia, a hot climate country, and to compare the average readings with data from previous studies. METHODS: Thirty healthy young adults (13 males and 17 females) who ranged in age from 20 to 40 years (mean±SD: 28.57±5.58 years) with no symptoms of dry eyes or ocular disease, who did not wear contact lenses, and were not pregnant or breastfeeding were enrolled in the study. For inclusion criteria, all subjects were examined under a slit-lamp to verify the absence of ocular surface disorders. Moreover, phenol red thread, tear breakup time (TBUT) tests, and McMonnies questionnaire were used to exclude dry eyes. Three measurements of tear osmolarity were performed in 1 eye (at 5-min intervals) using the TearLab osmometer. RESULTS: The mean score on the McMonnies questionnaire was 6±2.9. The mean values obtained from the phenol red thread and TBUT tests were 22±7.7 mm (OD), 21.5±6.6 mm (OS) and 12±1.7 s (OD), 12.5±1.6 s (OS), respectively. There were no statistically significant differences between the osmolarity readings (Wilks' lambda; P=0.945). Tear osmolarity results confirmed that 50% of subjects had readings of 299 to 312 mOsm/L, 40% had readings of 288 to 299 mOsm/L, and 10% had readings of 277 to 288 mOsm/L. The overall mean osmolarity was 299.06±7.65 mOsm/L. There was a medium correlation between osmolarity and the McMonnies questionnaire and a strong negative correlation between osmolarity and TBUT values. CONCLUSIONS: We found that the average osmolarity for subjects living in Saudi Arabia was 299.06±7.65 mOsm/L, which falls within the reference range and is in agreement with published results (298.0-301.9±7.2-16.0 mOsm/L). Osmolarity was positively correlated with McMonnies questionnaire and negatively correlated with TBUT values. The TearLab osmolarity system test showed good precision in repeated measurements. This is the first report of use of the TearLab osmolarity system to assess tear film in normal Saudi adults.

Keratoconus corneal architecture after riboflavin/ultraviolet A cross-linking: ultrastructural studies.

PURPOSE: Study to investigate the effects of collagen cross-linking on the ultrastructural organization of the corneal stroma in the human keratoconus cornea (KC). METHODS: Three normal, three keratoconus (KC1, KC2, KC3), and three cross-linked keratoconus (CXL1, CXL2, CXL3) corneas were analyzed. The KC corneas were treated with a riboflavin-ultraviolet A (UVA) treatment (CXL) method described by Wollensak et al. Penetrating keratoplasty (PKP) was performed 6 months after treatment. All samples were processed for electron microscopy. RESULTS: The riboflavin-UVA-treated CXL corneal stroma showed interlacing lamellae in the anterior stroma followed by well-organized parallel running lamellae. The lamellae contained uniformly distributed collagen fibrils (CFs) decorated with normal proteoglycans (PGs). The CF diameter and interfibrillar spacing in the CXL cornea were significantly increased compared to those in the KC cornea. The PG area in the CXL corneas were significantly smaller than the PGs in the KC cornea. The epithelium and Bowman's layer were also normal. On rare occasions, a thick basement membrane and collagenous pannus were also observed. CONCLUSIONS: Corneal cross-linking leads to modifications of the cornea stroma. The KC corneal structure showed a modification in the CF diameter, interfibrillar spacing, and PG area. This resulted in a more uniform distribution of collagen fibrils, a key feature for corneal transparency.

Ultrastructure features of camel cornea--collagen fibril and proteoglycans.

INTRODUCTION: The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs). METHODS: Camel corneas (of 6-, 8-, and 10-month-old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The 'AnalySIS LS Professional' program was used to analyze the collagen fibril diameter. RESULTS: The camel cornea consists of four layers: the epithelium (227 µm), stroma (388 µm), Descemet's membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 µm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM. CONCLUSION: The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.

Lipopolysaccharide Enhances Genotoxicity by Activating GADD45G and NF-κB in Human Corneal Epithelial Cells.

As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 µg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 µg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.

Measurements of Tear Evaporation Rate in Subjects with Refractive Errors Using a Portable Evaporimeter.

Dry eye symptoms are associated with refractive errors. We aimed to measure the tear evaporation rate (TER) in subjects with refractive errors (RE) using a portable evaporimeter. This nonrandomized, case-control, and observational study included 75 subjects, including 25 subjects aged 18-38 years (28.8 ± 6.8 years) with myopia (-0.75 to -3.75 D) and 25 subjects aged 18-39 years (27.7 ± 5.5 years) with hyperopia (+0.50 to +3.75 D). In addition, a control group of 25 subjects with emmetropic eyes aged 20-30 years (23.6 ± 2.6 years) was recruited. The ocular surface disease index (OSDI) was completed, followed by the TER measurements using a portable evaporimeter. The OSDI score and TER measurements showed dry eye symptoms in 36% and 48% of myopic subjects, respectively. For hyperopic participants, dry eye was observed in 24% and 56% of the subjects based on the OSDI and TER scores, respectively. Significant differences (Mann-Whitney U test; p < 0.001) were found among the OSDI and TER scores recorded within the study (myopic and hyperopic subjects) and control groups. Significant strong positive correlations were detected between the OSDI and TER scores in both myopic (p = 0.004; r = 0.559) and hyperopic (p = 0.001; r = 0.619) subjects. The TER scores were significantly higher in subjects with RE (myopic and hyperopic) as compared with individuals with normal eyes.

Improving tear ferning patterns collected from goats and camels after adding various electrolyte solutions.

BACKGROUND: Good quality of tear film is essential for healthy vision in both animals and humans. Therefore, improving the quality of tears through the addition of electrolytes is important. OBJECTIVES: To assess the effect of adding various electrolyte solutions on tear ferning (TF) patterns collected from goats and camels. MATERIAL AND METHODS: Tear samples (20 ƒÊL) were collected from 5 goats (2 males and 3 females; 3.4 }1.6 years) and 5 camels (2 males and 3 females; 4.0 }1.1 years) using microcapillary tubes. A tear sample (0.5 ƒÊL) from each animal was mixed with various volumes (0.5.5 ƒÊL) of each electrolyte solution to produce homogenous mixtures. A sample (1 ƒÊL) of each mixture was dried on a microscopic glass at 22°C with a humidity .40%. The obtained TF pattern was observed, graded and compared with those obtained for the corresponding pure tear samples. The effect of dilution using purified water on the TF patterns of animals was also tested. RESULTS: The TF grades of animals were generally enhanced when mixed with electrolyte solutions. Specifically, the TF grade for tears collected from a goat was improved from 1.4 to 0.7 and to 0.8 when magnesium chloride hexahydrate and calcium chloride were added, respectively. Similarly, the TF grade for tears collected from a camel was improved from 1.8 to 0.9 and to 1.1, when calcium chloride and sodium dihydrogen phosphate solutions were added, respectively. CONCLUSIONS: The TF grades of tears collected from both goats and camels were improved after adding electrolyte solutions, and they were most remarkably improved when divalent electrolyte solutions were added, followed by the hydrogenated electrolyte.

A unique pre-endothelial layer at the posterior peripheral cornea: ultrastructural study.

This study was conducted to investigate the ultrastructure of a unique structures at the anterior side of the endothelium of the posterior peripheral cornea and compare their inner fibers to those of the limbus and sclera. The unique structures at the anterior side of endothelium was referred as a pre-endothelial (PENL) structures in the present manuscript. Ten anonymous-donor human corneoscleral rims (leftover after corneal transplants) were processed for electron microscopy. Semi-thin sections were examined using an Olympus BX53 microscope, and ultrathin sections were studied using a JOEL 1400 transmission electron microscope. A unique PENL structures was identified at the posterior peripheral cornea at a radial distance of approximately 70-638 µm, from the endpoint of Descemet's membrane. The PENL thinned out gradually and disappeared in the center. The contained an electron-dense sheath with periodic structures (narrow-spacing fibers), wide-spacing fibers, and numerous microfibrils. Typical elastic fibers were present in the sclera and limbus but were not observed in the PENL. This study revealed the existence of a new acellular PENL, containing unique fibrillar structures that were unseen in the corneal stroma. From the evidence describe in this paper we therefore suggest that PENL is a distinct morphological structure present at the corneal periphery.

Structure of corneal layers, collagen fibrils, and proteoglycans of tree shrew cornea.

PURPOSE: The stroma is the major part of the cornea, in which collagen fibrils and proteoglycans are distributed uniformly. We describe the ultrastructure of corneal layers, collagen fibrils (CF), and proteoglycans (PGs) in the tree shrew cornea. METHODS: Tree shrew corneas (5, 6, and 10 week old animals) and normal human corneas (24, 25, and 54 years old) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in a sodium acetate buffer. The tissue was processed for electron microscopy. The 'iTEM Olympus Soft Imaging Solutions GmbH' program was used to measure the corneal layers, collagen fibril diameters and proteoglycan areas. RESULTS: The tree shrew cornea consists of 5 layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. The epithelium was composed of squamous cells, wing cells and basal cells. The Bowman's layer was 5.5±1.0 µm thick and very similar to a normal human Bowman's layer. The stroma was 258±7.00 µm thick and consisted of collagen fibril lamellae. The lamellae were interlaced with one another in the anterior stroma, but ran parallel to one another in the middle and posterior stroma. Collagen fibrils were decorated with proteoglycan filaments with an area size of 390 ±438 nm(2). The collagen fibril had a minimum diameter of 39±4.25 nm. The interfibrillar spacing was 52.91±6.07 nm. Within the collagen fibrils, very small electron-dense particles were present. CONCLUSIONS: The structure of the tree shrew cornea is very similar to that of the normal human cornea. As is the case with the human cornea, the tree shrew cornea had a Bowman's layer, lamellar interlacing in the anterior stroma and electron-dense particles within the collagen fibrils. The similarities of the tree shrew cornea with the human cornea suggest that it could be a good structural model to use when studying changes in collagen fibrils and proteoglycans in non-genetic corneal diseases, such as ectasia caused after LASIK (laser-assisted in situ keratomileusis).

Ultrastructural study of collagen fibrils, proteoglycans and lamellae of the cornea treated with iontophoresis - UVA cross-linking and hypotonic riboflavin solution.

To investigate the effects of iontophoresis-ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1) , a UVA irradiance of 3 mW/cm2 for 30 min; Group 2 (G2) , a UVA irradiance of 10 mW/cm2 for 9 min; Group 3 (G3) , without UVA irradiation. Three untreated corneas were used as controls ( G0 ). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm2 for 30 min and 10 mW/cm2 for 9 min was a suitable method of CXL; however, 3 mW/cm2 for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.

Improvement in Tear Ferning Patterns of Sheep Tears After Addition of Various Electrolyte Solutions.

Objective: This study aimed to improve the tear ferning (TF) patterns in the sheep tears after the addition of various electrolyte solutions in different proportions. Animal Studied: Sheep were located at a small farm in the outskirts of Riyadh, Saudi Arabia. The sheep had no ocular disorders or diseases, and none of the female sheep were pregnant. Methods: Tear samples (20 µl) were collected from the right eyes of seven healthy sheep (five female sheep and two male sheep; age 7-36 months with an average of 17.0 ± 10.3 months). A tear sample (1 µl) from each sheep was dried on a microscopic glass slide at 22°C and <40% humidity. The TF patterns were graded based on the five-point grading scale in 0.1 increments. Homogenous mixtures were prepared by mixing tears from each sheep (0.5 µl) with various electrolyte solutions in different proportions (1:1, 1:2, 1:4, 1:6, 1:8, and 1:10). A sample of each mixture (1 µl) was dried on a glass slide, and the TF patterns for each mixture were observed, recorded, graded, and compared with those of the corresponding pure sheep tears. In addition, each sheep tear sample (0.5 µl) was diluted with pure water (0.5 µl) and the TF images were recorded and graded to test the dilution effect. Results: General improvement was noted in TF grades after the addition of electrolyte solutions, ranging from 1.7-1.4 to 1.3-0.3 regardless of the ratio between the electrolyte solutions and sheep tears within the mixture. TF grades of sheep tear samples improved significantly after adding different volumes of calcium chloride solution. Similar improvements in TF grades were observed when magnesium chloride hexahydrate and sodium dihydrogen phosphate solutions were used as the electrolytes. Some improvements in the TF grades occurred with the addition of potassium chloride to sheep tear samples. There was little improvement in TF grades after the addition of sodium chloride solution. Conclusion: Tear ferning grades of sheep tear samples improved when mixed with a number of electrolyte solutions at different volumes, in particular with calcium chloride or magnesium chloride solutions. Some improvements in TF grades were seen with sodium dihydrogen phosphate or potassium chloride solution added as the electrolyte. Clearly, divalent electrolytes lead to a greater improvement in TF grades of sheep tear samples as compared with sodium dihydrogen phosphate or monovalent electrolytes.