Sagrario Callejo

Characterization of epithelial primary cultures from human conjunctiva

Primary cultures of human epithelial cells from normal conjunctiva were developed and characterized to determine whether they retained epithelial characteristics. Conjunctival explants were obtained from the upper fornix of healthy donors and cultured in supplemented DMEM/F-12 medium for 5 days. The epithelial outgrowth was maintained for an additional 10 days. Primary cultures were then processed for light microscopy, transmission and scanning electron microscopy (TEM, SEM), and immunocytochemistry. They exhibited typical features of conjunctival epithelium on light microscopy (polygonal morphology, intimate cohesion, production of mucins), TEM (abundant desmosomes, keratin bundles, granules, microvilli), SEM (polygonal shape, microvilli, intimate cohesion), and immunocytochemistry (positivity for the receptor of epidermal growth factor, desmosomal proteins, and cytokeratins). In conclusion, primary cultures developed from normal human conjunctiva maintained the epithelial characteristics in vitro. Because the conjunctiva is a major component of the anterior ocular surface, we propose this in vitro system as suitable for physiopathologic and toxicologic studies.

CARBOMER- VERSUS CELLULOSE-BASED ARTIFICIAL-TEAR FORMULATIONS : MORPHOLOGIC AND TOXICOLOGIC EFFECTS ON A CORNEAL CELL LINE

PURPOSE Frequent instillation of artificial tears is the primary disadvantage of the treatment for dry-eye syndrome. Recently gel formulations have been proposed as an alternative to classic cellulose formulations. The higher viscosity of these gels presumably prolongs tear-retention time in the eye and results in fewer daily applications. However, no toxicologic studies with gel formulations have been performed. Our aim was to study the toxic effects of these formulations on corneal cells. METHODS SIRC cells from rabbit cornea were exposed for 30 min, and 1, 3, and 6 h to five commercially available artificial tears, three of them carbomer gel formulations (Lacrivisc, Lacrivisc unit-doses, and Viscotears) and two carboxymethylcellulose (CMC) formulations (Celluvisc and Cellufresh). A cytotoxicity assay and a scanning electron microscopy (SEM) study were used to analyze the putative toxic effects of the formulations. The preservatives of the gel formulations also were tested. RESULTS Carbomer gel formulations, both with and without preservatives, caused more in vitro toxic effects in the corneal cells than did CMC formulations and caused severe damage even after 30 min of exposure. SEM revealed dramatic cell-surface alterations. Preservatives added to Lacrivisc and Viscotears also had toxic effects on cells, whose effects were not significantly different from those of the commercial preparations. CONCLUSION These results demonstrate that in the in vitro study, CMC artificial tears are less toxic than carbomer gel formulations. Questions about the benefits of using high-viscosity gels in the treatment of dry-eye syndrome still remain.

Human Conjunctival Epithelium in Culture: A Tool to Assay New Therapeutic Strategies for Dry Eye

Conjunctival changes in dry eye syndrome involve alteration in the mucous layer of the tear film. Mucus production and secretion is one of the principal roles of the conjunctival epithelium. Mucins, the main components of the mucous layer, are glycoproteins produced by goblet and non-goblet epithelial cells from the conjunctiva1–3 and by corneal epithelial cells.4 Any process that interferes with normal mucin production also interferes with proper wetting of the ocular surface.

Focal adhesion kinase as a mechanotransducer during rapid brain growth of the chick embryo.

Expansion of the hollow fluid-filled embryonic brain occurs by an increase in intraluminal pressure created by accumulation of cerebrospinal fluid (CSF). Experiments have shown a direct correlation between cavity pressure and cell proliferation within the neuroepithelium. These findings lead us to ask how mechanistically this might come about. Are there perhaps molecules on the luminal surface of the embryonic neuroepithelium, such as focal adhesion kinases (FAKs) known to respond to tension in other epithelial cells? Immunodetection using antibodies to total FAK and p-FAK was performed with subsequent confocal analysis of the pattern of their activation under normal intraluminal pressure and induced chronic pressure. Western analysis was also done to look at the amount of FAK expression, as well as its activation under these same conditions. Using immunolocalization, we have shown that FAK is present and activated on both apical and basolateral surfaces and within the cytoplasm of the neuroepithelial cells. This pattern changed profoundly when the neuroepithelium was under pressure. By Western blot, we have shown that FAK was upregulated and activated in the neuroepithelium of the embryos just after the neural tube becomes a closed pressurized system, with phosphorylation detected on the luminal instead of the basal surface, along with an increase in cell proliferation. Chronic hyper-pressure does not induce an increase in phosphorylation of FAK. In conclusion, here we show that neuroepithelial cells respond to intraluminal pressure via FAK phosphorylation on the luminal surface.

α2-Adrenergic Receptors Are Present in Normal Human Conjunctiva

Purpose: Despite the passage of medications, including antiglaucoma drugs, through the ocular surface, and despite the increasing relevance of neurogenic inflammation in the ocular surface, the presence of some neuroreceptors in the conjunctiva has not been ascertained. This study describes the presence of α2-adrenergic receptors in normal human conjunctiva. Methods: Immunofluorescence microscopy, electrophoresis, and Western blot analyses were done in human conjunctival biopsies and rat control tissues. Antibodies against α2-adrenergic receptor subtypes α2A, α2B, and α2C were used. Results: Immunoreactivity for α2A- and α2B-, but not α2C-adrenergic receptors was evenly distributed in epithelial cells of human conjunctiva cryosections. Immunoreactive bands were detected for the three α2-adrenergic receptor subtypes: a major band of 48–50 kDa and fellow bands of 65–67 kDa. Conclusions: Normal human conjunctival epithelial cells express α2A-, α2B-, and α2C-adrenergic receptors. Further studies to determine the functional implications of these receptors in ocular surface homeostasis are warranted.