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.

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.

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.

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.

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.

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.

Inhibitory Effect of Ursolic Acid on Ultraviolet B Radiation-Induced Oxidative Stress and Proinflammatory Response-Mediated Senescence in Human Skin Dermal Fibroblasts.

Ultraviolet radiation is an environmental carcinogenic agent that enhances inflammation and immunological reactions in the exposed human skin cells leading to oxidative photoaging of the epidermal and dermal segment. In the present study, we investigated the protective role of ursolic acid (UA) against ultraviolet B (UVB) radiation- induced photoaging an in vitro model of human skin dermal fibroblasts. UA-pretreated human skin dermal fibroblast (HDF) cells were exposed to UVB radiation to evaluated cell viability, reactive oxygen species (ROS), mitochondrial membrane potential, lipid peroxidation, antioxidant status, DNA damage, proinflammatory response, apoptotic induction, and matrix metalloproteinase (MMP) alteration. The UA pretreatment of HDFs mitigated the UVB irradiation-induced cytotoxicity, ROS generation, and mitochondrial membrane potential alteration and lipid peroxidation, depletion of antioxidant status, DNA damage, and apoptotic induction. UA pretreatment of HDFs also attenuated the UVB-induced expression of inflammatory (TNF-α and NF-κB) and apoptotic (p53, Bax, and caspase-3) and MMPs (MMP-2 and MMP-9) and enhanced the Bcl-2 protein levels in 20 µM UA treatment, when compared to concentrations. Hence, these results revealed that UA has the potential to mitigate UVB-induced extracellular damage by interfering with the ROS-mediated apoptotic induction and photoaging senescence and thus is a potential therapeutic agent to protect the skin against UVB-irradiation induced photooxidative damage.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography.

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet's membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Clinical and Ultrastructural Studies of Gelatinous Drop-Like Corneal Dystrophy (GDLD) of a Patient with TACSTD2 Gene Mutation.

PURPOSE: To describe clinical, molecular genetics, histopathologic and ultrastructural findings of gelatinous drop-like corneal dystrophy (GDLD) (OMIM #204870) in a Sudanese patient. METHOD: An ocular examination revealed the onset of GDLD in a Sudanese patient (50 years old) at King Khalid Specialist Hospital, Riyadh. The 333 sequence variants in 13 GDLD genes of a DNA sample were screened by Asper Ophthalmics Ltd. It was further confirmed by sequencing. The patient had undergone a penetrating keratoplasty in the right eye. The corneal tissue was processed for histopathology and ultrastructural studies. RESULTS: Slit-lamp observation showed grayish-white multiple superficial corneal nodules of various sizes in the left and right eye. Both corneas became clear after the surgery. The GDLD deposits in the subepithelial region and in the anterior stroma were confirmed by PAS staining and their apple-green birefringence under polarized light. Ultrastructurally, the amyloid fibrils were very thin and grouped in whorl-like structures, which caused splits between and within the stromal lamellae. Collagen fibrils (CFs) and keratocytes had degenerated. A homozygous c.355T > A mutation in exon 1 of the TACSTD2 (M1S1) gene was detected, and alteration of the amino acid (p.Cysl19Ser in NCBI entry NP_002344.2) was observed. CONCLUSION: In our patient with GDLD, a "c.355T > A" mutation in exon 1 of TACSTD2 was detected and believed to be responsible for the alteration of the amino acid leading to the formation of the amyloid deposits. The deposits caused the ultrastructural degeneration of epithelium, Bowman's layer, stroma, and keratocytes of the GDLD cornea.

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.

Ultrastructural study of peripheral and central stroma of keratoconus cornea.

PURPOSE: Assess the lamellar organisation of the peripheral and central stroma of the keratoconus (KC) and normal cornea. METHODS: Five normal and three KC corneas were fixed in 2.5% glutaraldehyde and processed for electron microscopy. The ultrathin sections were observed under JEOL 1400 TEM, and digital images were taken with a bottom-mounted 11-megapixel Quamisa camera, using the iTEM software. Measurements of the lamellae were carried out using the iTEM software. Statistical analysis was performed using the SPSS software. RESULTS: The lamellar organisation at the centre and periphery of the KC cornea was disrupted by the presence of multiple undulations, which were more aggressive at the posterior stroma. Among the KC cornea, the mean lamellar thickness of the peripheral middle (1030.32±86.25 nm) and posterior (615.68±30.94 nm) stroma was also significantly (p<0.05) thinner than their corresponding areas of the central KC cornea (1151.1±48 nm; 783.57±31.10 nm). At the periphery of KC cornea, just above the Descemet's membrane (DM), small undulations appeared to emerge out from the DM. Furthermore, the anterior stroma of the peripheral cornea contained several lamellar sutures. The mean lamellar thickness of the peripheral and central KC cornea was significantly (p<0.0001) thinner than the corresponding areas of the normal cornea. CONCLUSION: The present study reveals the involvement of lamellae in the peripheral stroma in the pathogenicity of the KC cornea. The emergence of small undulations in the DM suggests that the formation of undulation might be starting from the DM.

Cornea and its adaptation to environment and accommodation function in veiled chameleon (Chamaeleo calyptratus): ultrastructure and 3D transmission electron tomography.

We investigate the ultrastructural features and 3D electron tomography of chameleon (Chamaeleon calyptratus) which is a native of desert environments of Saudi Arabia. The corneas of the chameleon were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for electron microscopy and tomography, and observed under a JEOL 1400 transmission electron microscope. The thin cornea (21.92 µm) contained 28-30 collagen fibril lamellae. The middle stromal lamellae (from 13 to 19) contained keratocytes with a long cell process and filled with granular material. The CF diameter increased from lamella 1 (30.44 ± 1.03) to Lamella 5 (52.83 ± 2.00) then decreased towards the posterior stoma. The percentage of large CF diameters (55-65 nm) was very high in the lamellae L14 (38.8%) and L15 (85.7%). The mean PGs area of the posterior stroma (448.21 ± 24.84 nm2 ) was significantly larger than the mean PGs area of the anterior, (309.86 ± 8.2 nm2 ) and middle stroma 245.94 ± 8.28 nm2 ). 3D electron tomography showed the distribution of PGs around and over the CF. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. This accommodation function is achieved by bending of the cornea. During bending the anterior stroma was stretched and the posterior stroma was compressed due to the presence of small CFs. The middle stroma remained stiff due to the presence of large CFs. Large proteoglycans not only maintain hydration for a longer period of time, but also act as a lubricant to neutralise the shear forces in the anterior and posterior stroma during bending.

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.

Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma.

We report here the ultrastructural organization of collagen fibrils (CF) and proteoglycans (PGs) of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS)/dermatan sulfate (DS) PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark.

Effectiveness of in-office blood pressure measurement by eye care practitioners in early detection and management of hypertension.

AIM: To investigate the number of hypertensive patients, the optometrist is able to identify by routinely taking blood pressure (BP) measurements for patients in "at-risk" groups, and to sample patients' opinions regarding in-office BP measurement. Many of the optometrists in Saudi Arabia practice in optical stores. These stores are wide spread, easily accessible and seldom need appointments. The expanding role of the optometrist as a primary health care provider (PHCP) and the increasing global prevalence of hypertension, highlight the need for an integrated approach towards detecting and monitoring hypertension. METHODS: Automated BP measurements were made twice (during the same session) at five selected optometry practices using a validated BP monitor (Omron M6) to assess the number of patients with high BP (HBP) -in at-risk groups-visiting the eye clinic routinely. Prior to data collection, practitioners underwent a two-day training workshop by a cardiologist on hypertension and how to obtain accurate BP readings. A protocol for BP measurement was distributed and retained in all participating clinics. The general attitude towards cardiovascular health of 480 patients aged 37.2 (±12.4)y and their opinion towards in-office BP measurement was assessed using a self-administered questionnaire. RESULTS: A response rate of 83.6% was obtained for the survey. Ninety-three of the 443 patients (21.0%) tested for BP in this study had HBP. Of these, (62 subjects) 66.7% were unaware of their HBP status. Thirty of the 105 subjects (28.6%) who had previously been diagnosed with HBP, still had HBP at the time of this study, and only 22 (73.3%) of these patients were on medication. Also, only 25% of the diagnosed hypertensive patients owned a BP monitor. CONCLUSION: Taking BP measurements in optometry practices, we were able to identify one previously undiagnosed patient with HBP for every 8 adults tested. We also identified 30 of 105 previously diagnosed patients whose BP was poorly controlled, twenty-two of whom were on medication. The patients who participated in this study were positively disposed toward the routine measurement of BP by optometrists.