Abstract
Purpose :
There is a need to develop new ocular surface (OS) models alternative to the use of living animals. Porcine eye is one of the closest to human but there is scarce knowledge of its OS. Our aim was to describe the porcine OS and study its ex vivo functionality as a model of corneal barrier to test drug penetration.
Methods :
Eyes and eyelids of 4 adult domestic pigs (Sus scrofa domestica) were exenterated immediately after sacrifice in an abattoir and fixed in 4% formalin. Tissues were embedded in paraffin and sectioned (5 μm). Slides were stained with alcian blue-periodic acid Schiff and analyzed using the Automated Cellular Imaging System III. Acidic, basic, and mixed conjunctival goblet cells (GC) were manually counted. To test drug penetration, eyeballs were placed in 6-well plates and submerged in PBS up to the limbus. A silicon ring was placed over the cornea and the fluorescent molecule Nile Red administered for 30 min as oil solution (control) or loaded in nanoparticles. Then, tissues were processed for fluorescence microscopy evaluation.
Results :
Porcine OS was similar to that of humans, although pigs have a third eyelid and thicker corneas than humans. We observed 6-7 corneal epithelial layers including 1-2 cuboidal, 3-4 wing and 1-2 squamous cell layers, while the limbus showed up to 12 epithelial cell layers. The conjunctiva showed 3 anatomical zones: palpebral, bulbar and fornix, with 6-8, 6 and 4 epithelial cell layers, respectively. Mostly individual GC were identified in upper eyelid (29%), third eyelid (35%), lower eyelid (16%), fornix (19%) and bulbar conjunctiva (1%). The third eyelid had 44% of total palpebral GC. GC contents were a mixture of acidic+basic glycoconjugates in 84% of cells. Abundant glands including Meibomian glands identified in eyelids were distributed similarly as in human eyelids. No fluorescence appeared in Nile Red solution-exposed corneas while intense fluorescence was observed through porcine corneas exposed to Nile Red-loaded nanoparticles.
Conclusions :
Similarities found between pig and human OS regarding structure, cellular population and glandular distribution make porcine tissues useful to test drug penetration in addition to other uses. Difficulties to access human tissue, misidentification and cross-contamination of cell lines, and the need to reduce the use of experimental animals further support the use of alternative tissue sources such as pig eyes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.