Maria Fernanda Suarez

Pro-Inflammatory Cytokines and Gelatinases in Climatic Droplet Keratopathy

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.

On the Cornea of Healthy Merino Sheep: A Detailed Ex Vivo Confocal, Histological and Ultrastructural Study

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.

Climatic droplet keratopathy: an old disease in new clothes

Climatic droplet keratopathy (CDK) is an acquired and potentially handicapping cornea degenerative disease that is highly prevalent in certain rural communities around the world. It predominantly affects males over their forties. It has many other names such as Biettis band‐shaped nodular dystrophy, Labrador keratopathy, spheroidal degeneration, chronic actinic keratopathy, oil droplet degeneration, elastoid degeneration and keratinoid corneal degeneration. CDK is characterized by the haziness and opalescence of the corneas most anterior layers which go through three stages with increasing severity. Globular deposits of different sizes may be histopathologically observed under the corneal epithelium by means of light and electron microscopy. The coalescence and increased volume of these spherules may cause the disruption of Bowmans membrane and the elevation and thinning of the corneal epithelium. The exact aetiology and pathogenesis of CDK are unknown, but they are possibly multifactorial. The only treatment in CDK advanced cases is a corneal transplantation, which in different impoverished regions of the world is not an available option. Many years ago, the clinical and histological aspects of this disease were described in several articles. This review highlights new scientific evidence of the expanding knowledge on CDKs pathogenesis which will open the prospect for new therapeutic interventions.

Genetic Background and Climatic Droplet Keratopathy Incidence in a Mapuche Population from Argentina

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.

Climatic Droplet Keratopathy in Argentina: Involvement of Environmental Agents in Its Genesis Which Would Open the Prospect for New Therapeutic Interventions

Climatic droplet keratopathy (CDK) is a degenerative corneal disease of unknown etiology. We described CDK for the first time in Latin America in the Argentinean Patagonia (El Cuy). A deeper knowledge of CDK pathogenic mechanisms will provide new therapeutic strategies. For that reason we investigated the prevalence of CDK in El Cuy and its existence in other 3 provinces with similar climate. Patients eyes were examined, habits throughout lives were inquired about, and serum ascorbate (sAA) was determined. All individuals work outdoors for most of the day. All regions had normal O3 levels. Individuals from regions 1, 2, and 3 had very low consumption of vegetables/fruits and low sAA levels. Conversely, region 4 individuals had balanced diet and higher sAA concentrations. CDK was only found in region 3 where individuals had partial deficiency of sAA and did not use eye protection. No CDK was found in regions 1 and 2 where individuals had similar work activities and dietary habits to those in region 3 but wear eye protection. No disease was found in region 4 where individuals work outdoors, have balanced diet, and use eye protection. To summarize, the CDK existence was related not only to climate but also to the dietary habits and lack of protection from sunlight.

Efectos producidos por diferentes tipos de láser en córnea de cobayos: identificación de un láser capaz de provocar lesiones superficiales sin dejar cicatrices

PURPOSE Climatic droplets keratopathy (CDK) is closely associated with superficial corneal erosions and lack of protective mechanisms against the harmful effects of ultraviolet radiation (UVR) during a prolonged period of time. One of the difficulties in studying the pathogenic mechanisms involved in this human disease is the lack of an experimental animal model. In this paper, a study is conducted on the effects of 4 types of lasers at various powers and time conditions on the normal guinea pig corneas in order to select only one laser condition that reversibly injures the epithelium and superficial stroma, without leaving scarring. METHODS Damage was induced in the cornea of Guinea pigs using different powers and exposure times of 4 types of laser: argon, CO2, diode and Nd-Yag, and any injuries were evaluated by biomicroscopy (BM) and optical microscopy. Corneas from other normal animals were exposed to argon laser (350 mW, 0.3s, 50 μm of diameter), and the induced alterations were studied at different times using BM, optical coherence tomography (OCT) and transmission electron microscopy (TEM). RESULTS Only argon laser at 350 mW, 0.3s, 50 μm of diameter produced epithelium and superficial stroma lesions. Some leukomas were observed by BM, and they disappeared by day 15. Corneal thickness measured by OCT decreased in the eyes treated with argon laser during the first week. Using TEM, different ultra structural alterations in corneal epithelium and stroma were observed during the early days, which disappeared by day 15. CONCLUSIONS It was possible to develop reproducible corneal epithelium and anterior stroma injuries using Argon laser at 350 mW, 0.3s, 50 μm of diameter. In vivo and in vitro studies showed that injured corneas with these laser conditions did not leave irreversible microscopic or ultra structural alterations. This protocol of corneal erosion combined with exposure to UVR and partial deficiency of ascorbate in the diets of the animals for an extended period of time has been used in order to try to develop an experimental model of CDK.

Vitamin C Functions in the Cornea: Ultrastructural Features in Ascorbate Deficiency

The cornea plays important roles in the eye since it not only maintains the ocular pressure and protects against different aggressions and forces but constitutes the primary lens in many animals’ eyes as well. In order to fulfill these extremely crucial functions, the cornea requires sophisticated structures called the epithelium, Bowman’s membrane, the stroma, Descement’s layer, and the endothelium. The complexity of this tissue is such that despite many efforts, it has not been possible to obtain clinically viable corneas in the laboratory yet. Vitamin C or L-ascorbic acid is an essential vitamin and a water-soluble important nutrient many animals chemically synthesize from glucose by means of enzymatic reactions, with L-gulono-γ-lactone oxidase being the last step. Among the multiple roles played by this vitamin, its primary functions are to act as a cofactor for reactions requiring reduced iron or copper metalloenzyme and as a protective antioxidant that operates in the aqueous phases, both intra- and extracellularly. Ocular tissues, particularly the cornea, concentrate vitamin C, which might suggest that the cornea’s different structures need this vitamin to fulfill their roles. In this work, we present scientific evidence of the critical roles that ascorbic acid performs in maintaining the functions and ultrastructures of the corneal epithelium, the stroma, and the endothelium.Abstract The cornea plays important roles in the eye since it not only maintains the ocular pressure and protects against different aggressions and forces but constitutes the primary lens in many animals’ eyes as well. In order to fulfill these extremely crucial functions, the cornea requires sophisticated structures called the epithelium, Bowman’s membrane, the stroma, Descement’s layer, and the endothelium. The complexity of this tissue is such that despite many efforts, it has not been possible to obtain clinically viable corneas in the laboratory yet. Vitamin C or L-ascorbic acid is an essential vitamin and a water-soluble important nutrient many animals chemically synthesize from glucose by means of enzymatic reactions, with L-gulono-γ-lactone oxidase being the last step. Among the multiple roles played by this vitamin, its primary functions are to act as a cofactor for reactions requiring reduced iron or copper metalloenzyme and as a protective antioxidant that operates in the aqueous phases, both intra- and extracellularly. Ocular tissues, particularly the cornea, concentrate vitamin C, which might suggest that the cornea’s different structures need this vitamin to fulfill their roles. In this work, we present scientific evidence of the critical roles that ascorbic acid performs in maintaining the functions and ultrastructures of the corneal epithelium, the stroma, and the endothelium.

Phospholipidomic Studies in Human Cornea From Climatic Droplet Keratopathy

Climatic droplet keratopathy (CDK) is an acquired degenerative disease predominantly affecting males over 40 years old. It results in progressive corneal opacities usually affecting both eyes. CDK is multifactorial and its etiology remains unknown. Our recent findings are consistent with CDK pathology being driven by environmental factors with oxidative stress playing an important role (e.g.,, contributing to lipid peroxidation) rather than climate factors. The changes in corneal lipid composition affected by environmental factors remain understudied. The purpose of this study was to systematically investigate phospholipids profile (phosphatidylcholine [PC] and phosphatidylserine [PS]) in corneas from CDK patients using tandem mass spectrometry. Samples from CDK areas and from non‐affected areas were obtained from patients diagnosed with CDK who underwent cataract surgery, were subjected to lipid extraction using a modified Bligh and Dyer method; protein concentrations were determined using the Bradfords method. Lipids were identified and subjected to ratiometric quantification using TSQ Quantum Access Max triple quadrupole mass spectrometer, using appropriate class specific lipid standards. All phospholipid classes showed lower total amounts in affected areas compared to control areas from CDKs corneas. Comparative profiles of two phospholipid classes (PC, PS) between CDK areas and control areas showed several common species between them. We also found a few unique lipids that were absent in CDK areas compared to controls and vice versa. Lower amount of phospholipids in CDK areas compared to control areas could be attributed to the lipid peroxidation in the affected corneal regions as a consequence of increased oxidative stress. J. Cell. Biochem. 118: 3920–3931, 2017.