Histological and immunohistochemical characterization of the porcine ocular surface.

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 µm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 µm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 µm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment.

Inflammation plays a key role in dry eye disease (DED) affecting millions of people worldwide. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used topically to act on the inflammatory component of DED, but their limited aqueous solubility raises formulation issues. The aim of this study was development and optimization of functional cationic nanoemulsions (NEs) for DED treatment, as a formulation approach to circumvent solubility problems, prolong drug residence at the ocular surface and stabilize the tear film. Ibuprofen was employed as the model NSAID, chitosan as the cationic agent, and lecithin as the anionic surfactant enabling chitosan incorporation. Moreover, lecithin is a mixture of phospholipids including phosphatidylcholine and phosphatidylethanolamine, two constituents of the natural tear film important for its stability. NEs were characterized in terms of droplet size, polydispersity index, zeta-potential, pH, viscosity, osmolarity, surface tension, entrapment efficiency, stability, sterilizability and in vitro release. NEs mucoadhesive properties were tested rheologically after mixing with mucin dispersion. Biocompatibility was assessed employing 3D HCE-T cell-based model and ex vivo model using porcine corneas. The results of our study pointed out the NE formulation with 0.05% (w/w) chitosan as the lead formulation with physicochemical properties adequate for ophthalmic application, mucoadhesive character and excellent biocompatibility.

Novel anti-inflammatory liposomal formulation for the pre-ocular tear film: In vitro and ex vivo functionality studies in corneal epithelial cells.

In ocular surface inflammatory diseases, such as dry eye disease, long-term symptom relief requires targeting the inflammation itself rather than treating only the surface-associated dryness with artificial tears. Therefore, we included an anti-inflammatory agent in an unpreserved liposome-based (LP) formulation used as artificial tears. Our aim was to characterize and study its in vitro and ex vivo cell uptake and functionality. Human corneal epithelial (HCE) cells were used to study MPA-LP-induced effects after 60 min of exposure, using blank LP and non-LP MPA formulations as controls. A fluorescent labeled LP formulation was used to determine uptake by HCE cells and localization in ex vivo porcine corneas. The LP formulation complied with the required physicochemical properties and had no cytotoxicity on HCE cells after 60 min of exposure. HCE cells showed LP-associated fluorescence at 24, 48, and 72 h after 60 min of exposure, and the LP-associated fluorescence was uniformly distributed throughout the porcine corneal epithelium immediately after 5 min of exposure. MPA-LP increased protein expression and nuclear translocation of progesterone receptor in comparison with controls as determined by Western blotting and immunofluorescence. Moreover, MPA-LP significantly reduced the cell proliferation rate and IL-6 and IL-8 production 48 h after the exposure period, as determined by the alamarBlue assay and ELISA, respectively. None of these effects were evident in blank LP-exposed cells and non-LP MPA formulation reduced only IL-6 production. Our results suggest that the LP-based formulation, used to replenish the lipids of the tear film, can be loaded with anti-inflammatory agents that can be delivered into the cells and activate specific drug receptors. These agents can reduce inflammatory cytokine production and may be effective in the treatment of inflammatory processes associated with ocular surface diseases.