Abstract
Purpose :
Determine the expression of axon guidance proteins in normal cornea and their recovery after cornea neuro-epithelium injury.
Methods :
The expression of axon guidance proteins belonging to the Semaphorins, Ephrins and Netrins families of proteins, and their associated receptors was evaluated by qPCR and immunofluorescent staining (IF) in mouse trigeminal ganglia and cornea tissue. Corneas were subjected to epithelium debridement and the recovery of gene expression was analyzed at 0, 1, 3, 5, 7 and 14 days post injury. For qPCR, RNA was purified from trigeminal ganglia and corneal epithelium using an RNeasy Kit and cDNA obtained with a high capacity Reverse Transcription Kit. Gene expression was evaluated using predesigned mouse TaqMan gene expression assays and samples were analyzed (n=3 per condition) using validated internal controls. Statistical analysis was performed to compare recovery of gene expression versus untreated controls using a Student t-test and p<0.05. For IF staining, tissues were harvested and snap frozen in OCT and cryosections incubated with antibodies against different members of axon guidance proteins and imaged under a fluorescent microscope. Images (n=3 per treatment) were processed with Axiovision software under the same setting conditions.
Results :
We found that all axon guidance proteins evaluated are expressed in the trigeminal ganglia and cornea epithelium. In the trigeminal ganglia all proteins were found in the cell body and few of them were expressed in the axons projecting to the cornea. The receptors that are usually associated with these proteins such as Neuropilins, Plexins and VEGFr were also present in both tissues with certain degree of variation. After corneal epithelium injury there was a fast recovery of axon guidance proteins gene expression. They initially colocalized with the basal cells in the epithelium and remained in the first two epithelial cell layers after two weeks post injury.
Conclusions :
Axon guidance proteins are crucial for axonal targeting in the eye. In the cornea they are highly expressed in the steady state and quickly upregulated after injury. Their trophic and neuroregenerative role in adult corneas is still an unresolved issue
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.