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Dry eye disease (DED) has a prevalence of 5-35% in the general population worldwide. Intrinsic factors such as age, sex steroids imbalance, autoimmune diseases, and extrinsic factors such as refractive surgery, allergy, use of contact lenses, and dry environment contribute to this disease. DED affects both the surface of the eye and the tear film. Therefore, chronic dry eye is characterized by increased osmolarity of the tear film, and it is associated with inflammation of the lacrimal gland and the surface of the eye (Yamaguchi, 2018). To treat DED, the current and most common option includes several daily applications of lubricant eye drops. Nevertheless, these eye drops usually lead to local side effects, including ocular allergies and blurred vision.
To study DED, the development of a robust dry eye model is essential to testing new treatments. Here, an ex vivo porcine model of dry eye was established by incubating porcine corneas at low humidity (LH). The model was characterized by histological damage evaluation, epithelial thickness, and relevant dry eye markers such as interleukin 1 beta (IL-1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), occludin and galectin- 3. In the dry eye model proposed, it was verified a rise in the secretion of IL-1β, along with a mRNA expression upregulation of NF-κB, occludin, and galectin-3. Furthermore, it was observed a higher apoptosis rate in the dry eye model in comparison to controls (Netto et al., 2022).
Next, eye drops that are currently used to treat DED, composed of hyaluronic acid and dexamethasone, were used to determine if they were therapeutically effective in the established porcine dry eye model. Hence, it was demonstrated that the low humidity-induced effects could be partially reversed with the use of hyaluronic acid eye drops and successfully reversed with treatment with dexamethasone. As a result, the dry eye model proposed simulates the in vivo condition and enables animal-free testing of dry eye treatments, as the porcine eyes came from a local abattoir, and the animals were euthanized for nutritional purposes (Netto et al., 2022).
The further step in this research was to evaluate if the DNA- Nanoparticles (NPs), developed initially to function as a drug delivery platform, would also behave as a treatment option for DED. For that, the ex vivo porcine dry eye model and an in vitro human corneal model were submitted to LH stress and treated with the NPs. Therefore, it was verified that the NPs attached to the stressed in vitro and ex vivo models up 24 h of incubation at LH, demonstrating their affinity to bind damaged tissue as well. Moreover, the NPs prevented apoptosis caused by LH at the porcine model and hindered mRNA expression upregulation of galectin-3. However, the exact mechanisms for an anti- inflammatory role should still be investigated in further research.
The last aim of this project was to improve a method of isolation and cultivation of corneal cells so that in vitro models can be established for the study of DED and other corneal diseases. For that, the outgrowth method was defined with the use of porcine corneas that came from a local abattoir. Small explants of the corneal limbus were isolated and cultivated in flasks in two different conditions: with the use of fetal bovine serum (FBS) and without it. It was verified that cultures cultivated with FBS were more numerous and were composed mainly of epithelial cells and other cell types, probably fibroblasts. As for the cultures incubated without FBS, the cells were predominantly epithelial cells and keratocytes. Therefore, cell isolation with the outgrowth method and the use of FBS can be used to study injury processes, while cell isolation and cultivation without FBS can be useful to develop an in vitro model similar to healthy corneal conditions (Netto et al., 2023).
In conclusion, important steps for the study of DED were developed with this project, allowing the expansion of knowledge about models for studying DED ex vivo and in vitro, and a possible new therapy for the disease was investigated. |
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