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Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians., (© 2014 Blackwell Verlag GmbH.)

Purpose: To determine whether the incidence of and susceptibility to climatic droplet keratopathy (CDK), an acquired, often bilateral degenerative corneal disease, is influenced by the genetic background of the individuals who exhibit the disorder., Methods: To determine whether the disease expression was influenced by the genetic ancestry of CDK cases in native Mapuche of the northwest area of Patagonia in Argentina, we examined mitochondrial DNA and Y-chromosome variation in 53 unrelated individuals. Twenty-nine of them were part of the CDK (patient) population, while 24 were part of the control group. The analysis revealed the maternal and paternal lineages that were present in the two study groups., Results: This analysis demonstrated that nearly all persons had a Native American mtDNA background, whereas 50% of the CDK group and 37% of the control group had Native American paternal ancestry, respectively. There was no significant difference in the frequencies of mtDNA haplogroups between the CDK patient and control groups. Although the Y-chromosome data revealed differences in specific haplogroup frequencies between these two groups, there was no statistically significant relationship between individual paternal genetic backgrounds and the incidence or stage of disease., Conclusions: These results indicate a lack of correlation between genetic ancestry as represented by haploid genetic systems and the incidence of CDK in Mapuche populations. In addition, the mtDNA appears to play less of a role in CDK expression than for other complex diseases linked to bioenergetic processes. However, further analysis of the mtDNA genome sequence and other genes involved in corneal function may reveal the more precise role that mitochondria play in the expression of CDK.

Purpose: Climatic droplet keratopathy (CDK) is a degenerative disease of the cornea with possible involvement from matrix metalloproteinases (MMPs). Therefore, the authors investigated histologic distribution, levels, and molecular forms of MMP-2 and MMP-9, as well as tear fluid levels of MMPs and cytokines in CDK patients. They additionally examined UV-B-irradiation effect on production of gelatinases and cytokines by human corneal epithelial (HCE) cell culture model., Methods: Tears were collected from 20 unrelated individuals (10 with CDK and 10 controls). CDK affected corneas were haematoxylin-eosin stained and the presence and distribution of MMP-2 and MMP-9 was examined using immunohistochemistry. Gelatinases and cytokine secretion was measured in tears and supernatants from UV-B-exposed HCEs by immunoblotting, gelatin zymography, and protein array, respectively., Results: MMP-2 and MMP-9 values were significantly higher in tears collected from CDK patients than healthy controls and were accompanied by pro-inflammatory cytokine secretion. Immunohistochemistry showed that MMP-2 was expressed at the basement membrane zone in both control and affected corneas, but also marked the edges of the granular CDK deposits; MMP-9 expression was restrained to basal layers of the epithelium and was markedly induced in CDK corneas. In HCE cells, UV-B increased gelatinase secretion, with a striking effect on MMP-9, and was preceded by pro-inflammatory cytokine release., Conclusions: The authors demonstrate that the corneal epithelium could participate in CDK development as a source of cytokines and gelatinases. Additionally, in HCE cells, UV-B- modulated cytokine and subsequent MMP secretion. Local inhibition of cytokine secretion and gelatinases may prevent CDK progression.

Purpose: Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP-2, MMP-9, MMP-8 and MMP-13 and their tissue inhibitors TIMP-1 and TIMP-2 in tear fluid of patients with CDK., Methods: Seventeen CDK patients and 10 controls living in Argentine Patagonia received a complete eye examination, and MMPs and TIMP-1/2 were determined by immunofluorometric assay (IFMA), gelatin zymography and quantitative Western immunoblot analysis in tear samples., Results: The MMPs were detected mostly in their latent forms. The levels of MMP-9 and MMP-2 were found to be significantly elevated in CDK patients, whereas latent and active MMP-8 levels were significantly enhanced in controls. There was no significant difference in the level of MMP-13. TIMPs were found as part of complexes, and the TIMP-1 levels were significantly lower in patients than controls., Conclusion: Elevated MMP-2 and MMP-9 levels have been implicated in the failure of corneal re-epithelialization, and enhanced MMP-2 and MMP-9 levels in CDK patients suggest that these MMPs may play a role in corneal scarring in CDK. Elevated levels of MMP-8 suggest a defensive role for this MMP in inflammatory reactions associated with recurring corneal traumas. Decreased expression of TIMP-1 in CDK patients suggest deficient antiproteolytic shield likely to render the corneas of CDK patients vulnerable to enhanced MMPs. Overall, these data suggest a mechanistic link between MMPs and TIMP-1 level in cornea and tears with corneal scarring in CDK., (© 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.)

Purpose: In the visually debilitating condition of climatic droplet keratopathy, corneal transparency is progressively lost. Although the precise cause of the disease and the mechanism by which it progresses are not known, a lifetime exposure to high solar radiation and a vitamin C-deficient diet may be involved in its development. This study examines the effect of dietary ascorbate levels and ultraviolet (UV)-B exposure on corneal stromal structure., Methods: Eight guinea pigs were divided into four treatment groups (A, B, C, and D). For 15 weeks, Groups A and C were fed an ascorbate-rich diet (2 mg/100 g bodyweight/day), while Groups B and D received an ascorbate-deficient diet (0.07 mg/100 g bodyweight/day). For the last 12 weeks of the study, Groups C and D also experienced chronic UVB exposure (0.12 J/cm² for 40 min/day). Following euthanasia, the corneas were enucleated and their stromal ultrastructure examined using X-ray scattering and electron microscopy., Results: UVB exposure resulted in an increased corneal thickness (p<0.001), but this was not accompanied by a widespread expansion of the collagen fibrillar array, and in the case of ascorbate-deficient animals, stromal thickening was associated with the compaction of collagen fibrils (p<0.01). Neither UVB exposure nor ascorbic acid deficiency caused any change in the average diameter or D-periodicity of the stromal collagen fibrils., Conclusions: UVB-induced changes in the corneal ultrastructure were most pronounced in animals fed an ascorbic acid-deficient diet. This suggests that ascorbic acid may play a vital role in protecting the corneal stroma from the harmful effects of UVB.

Purpose: To determine levels of Peptidyl arginine deiminase 2 (PAD2) and its product protein-bound citrulline in cadaver eyes that suffered from normal tension glaucoma (NTG) compared to primary open angle glaucoma (POAG), and controls., Methods: Western analysis, ELISA, and immunohistochemical analysis were performed with human tissues., Results: We report over expression of PAD2 and higher levels of its product protein-bound citrulline in the optic nerve of normal tension glaucoma patients (NTG)., Conclusions: This is the first report demonstrating that like in POAG, NTG also possesses elevated levels of both PAD2 and protein-bound citrulline.

Objective: To describe the functional and structural characteristics of the cornea in healthy Guinea pigs., Animals Studied: Healthy male and female pigmented and albino Guinea pigs (Caviaporcellus) aged 3-5 months old were used., Procedures: The animals' corneas underwent different in vivo studies including: slit-lamp biomicroscopy, fluorescein staining (FS), break-up time test (BUT), confocal microscopy and pachymetry. The corneas were also studied histopathologically with light microscopy, immunohistochemistry and transmission electron microscopy., Results: No significant differences were found between pigmented and albino animals, male and female, OD and OS in any study performed. The differences on corneal thickness values were not significant among central (227.85 +/- 14.09 microm) and upper and temporal peripheral regions (226.60 +/- 12.50 and 225.70 +/- 14.40 microm, respectively). All histological studies performed permitted identification and precise description of the different corneal structures in Guinea pigs: the stratified epithelium (45.52 +/- 5.26 microm), Bowman's layer (2.23 +/- 0.38 microm), stroma (163.69 +/- 4.90 microm), Descemet's membrane (3.96 +/- 0.46 microm) and the endothelium (5.09 +/- 0.71 microm). Combining results from all eyes mean and SD from corneal BUT values was 4.98 +/- 1.67 s. Corneas often showed discrete superficial erosions being the FS positive in both eyes from all the animals., Conclusion: This study provides a detailed in vivo and postfixed histological description of the Guinea pig's cornea and information about the physiological tests.

Glycoproteins are potentially important biomarkers of disease and therapeutic targets. In particular, the N-linked glycoproteins are a focus of interest as they can be found in the extracellular environment and body fluids. In this study, we have sampled the tears, the extracellular fluid of the epithelial cells covering the surface of the eye, of patients with climatic droplet keratopathy (CDK) using tears of unaffected normal patients for comparison. Prefractionation of the tear sample used a hydrazide-resin capture method, and the previously N-glycosylated peptides were then subjected to two-dimensional nano-LC-nano-ESI-MS/MS analysis to obtain peptide fragmentation patterns for identification through protein database searches. We have identified a total of 43 unique N-glycoproteins, 19 of which have not previously been reported in tear fluid. In addition, we have quantitatively compared N-glycoprotein profiles in tear fluid of patients with CDK to tears of nondiseased controls using glycopeptide capture, iTRAQ labeling and 2D nano-LC-nano-ESI-MS/MS analysis. In tears of CDK patients, increased levels of four N-glycosylated proteins including haptoglobin (at sites N207, N211 and N241), polymeric immunoglobulin receptor (at sites N83, N90, N135, N186, N421, and N469), immunoglobulin J chain (at site N49) and an uncharacterized protein DKFZp686M08189 (at site N470), as well as a decrease in the N-glycosylation level of one N-glycosylated protein, lacritin (at site N119) were observed. However, the overall levels of these five proteins showed no appreciable changes between control and CDK samples. The findings could be clinically significant in terms of disease etiology and biomarkers.

Purpose: To identify the proteins in the corneal droplets of climatic droplet keratopathy (CDK), a disease that results in the formation of droplets on the cornea. Progressive accumulation of droplets in CDK leads to visual loss., Methods: Proteomic mass spectrometry of the CDK specimens was performed after fractionation of proteins in 4% to 20% SDS-polyacrylamide gels. Droplets were derived from two human donors. Immunohistochemistry with antibodies was performed to confirm the presence of identified proteins on donor tissues from patients with CDK and control subjects., Results: Proteomic analyses revealed identification of 105 proteins in CDK specimens. Immunohistochemical analyses confirmed localization of annexin A2 and glyceraldehyde 3-dehydrogenase (GAPDH), proteins identified by proteomic analyses in CDK specimens. The proteins were subjected to analyses with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Database which showed that a few biochemical pathways were more frequent for the identified proteins., Conclusions: Approximately 105 proteins were identified in CDK specimens, and a subset of them was confirmed by immunohistochemistry. Several of these may play a role in fibril or deposit formation.