Abstract
Purpose :
To determine the functional effects of Netrin-4, a known modulator of axon guidance, tumorigenesis, and angiogenesis, on corneal epithelium and nerve regeneration.
Methods :
The endogenous expression of Netrin-4 was evaluated by immunofluorescent staining (IFS) in mouse cornea and trigeminal ganglia (TG) and human corneas. The recovery of Netrin-4 expression after corneal epithelium injury was confirmed by IFS and RT-qPCR. In vitro, human corneal limbal epithelial (HCLE) cells and freshly isolated TG neurons were treated with recombinant Netrin-4 in a dose-response manner. HCLE cells were cultured until confluency, and scratch assays were measured with time-lapse microscopy. TG neurons isolated from Thy1-YFP mice (N=3/treatment) were treated with medium only, NGF, or Netrin-4. Neuronal growth and neurite extension were then quantified with Neurolucida software. The in vivo effects of Netrin-4 were tested on mice (N=6/condition) using a corneal epithelial debridement model. Post-injury, mice were treated with topical drops of PBS or Netrin-4. Epithelial healing was evaluated with a slit lamp microscope and analyzed in Fiji. The corneas were flat mounted and stained on day 10 to assess subbasal nerve regeneration. Student’s t-test and ANOVA were used for statistical analyses.
Results :
Netrin-4 was highly expressed in the mouse and human corneal epithelium and the neuronal cell bodies of TG. After injury, Netrin-4 was initially downregulated in these tissues and slowly returned to baseline levels of expression. The scratch assays on HCLE cells show that Netrin-4 at 25ng/mL induced significantly faster wound closure than control. Similarly, the in vivo data showed that mice receiving topical Netrin-4 had accelerated wound healing compared to control mice. The average neurite length was greater in isolated TG neurons treated with 25ng/mL Netrin-4 (p=0.02) and 50ng/mL NGF (p<0.0001) compared to the control. In vivo, the average length of subbasal nerves was greater for mice treated with Netrin-4 than for those treated with vehicle.
Conclusions :
Netrin-4 was normally expressed in the corneal epithelium, and after injury, its expression slowly recovered to basal levels. Netrin-4 increased corneal epithelium and nerve regeneration both in vitro and in vivo. Ongoing studies aim to establish the optimal route of administration for Netrin-4 in corneal wound healing.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.