A new 3D model of the cornea — the eye’s outer layer — is life-like enough that it could be helpful for a variety of research uses, including screening multiple potential therapies for neurotrophic keratitis (NK) over a short time period.
The research was presented at the 2020 Association for Research in Vision and Ophthalmology Annual Meeting and is available as an abstract, entitled “Corneal nerve regeneration in novel 3D models vs in vivo approaches,” published in the journal Investigative Ophthalmology and Visual Science.
For this study, researchers from the Carl von Ossietzky University in Germany set out to understand why damaged corneal nerve plexus cells — a leading cause of NK — rarely completely heal.
Researchers believe that stimulating corneal nerve growth in an experimental setting might be a promising approach to treat NK. First, however, a better experimental model in which to monitor nerve growth is necessary. A good in vitro model allows researchers to rapidly perform early experiments, such as drug screens.
The team created a 3D model based on collagen gel to provide structure for growing nerves. They then embedded clusters of nerve cells called dorsal root ganglia from mouse spinal cords along the rim of the gel.
In the center, they implanted human corneal fibroblasts, cells important to wound repair and nerve growth. Finally, they placed limbal epithelial stem cells — important in maintaining eye structure — on top of the gel.
The team also created a model using only trigeminal nerve cells, which plays a key role in tear production, or dorsal root ganglia cells for comparison. A model containing more traditional 2D — cells grown on the surface of a petri dish — provided a final in vitro comparison.
The scientists incubated cells with a compound called Y27632, which blocks the action of Rho-associated kinase, or ROCK, a protein that limits nerve cell growth. They then measured changes in nerve growth at various time points.
Nerve cells in the 3D gel treated with Y27632 grew significantly longer than control cells — roughly 5.17 mm compared with 1.62 mm. The investigators observed comparable changes in the 2D models.
The experiment produced similar results in mice, providing in vivo support for the utility of the 3D gel-based model. Here, the researchers cut the corneal nerve plexus of the right eye and treated these eyes twice a day with either Y27632 or the liquid used to deliver Y27632, devoid of the active compound. They measured corneal nerve growth once a week for 28 days.
By day 28, treated corneal nerves had grown to 8.59 mm compared with 3.72 mm in the untreated eyes.
“This model seems to provide a suitable, in-vivo like environment for specific research questions, with the possibility to test a high number of different substances in a short period of time,” the authors concluded.