June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Netrin-4 modulates corneal wound healing by enhancing corneal epithelium and nerve regeneration in vitro and in vivo.
Author Affiliations & Notes
  • Cissy Xiao
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Daniel Lara
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Qiang Zhou
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Tara Thanh Nguyen
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Mark Rosenblatt
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Victor H Guaiquil
    Illinois Eye and Ear Infirmary, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   Cissy Xiao None; Daniel Lara None; Qiang Zhou None; Tara Nguyen None; Mark Rosenblatt None; Victor Guaiquil None
  • Footnotes
    Support  NIH R01EY027912 Grant to Mark I. Rosenblatt, RPB unrestricted Departmental grant, P30 EY001792 Core Grant for Vision Research and Eversight to Victor Guaiquil, University of Illinois at Chicago College of Liberal Arts and Sciences Undergraduate Research Initiative (LASURI)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3234 – A0269. doi:
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    • Get Citation

      Cissy Xiao, Daniel Lara, Qiang Zhou, Tara Thanh Nguyen, Mark Rosenblatt, Victor H Guaiquil; Netrin-4 modulates corneal wound healing by enhancing corneal epithelium and nerve regeneration in vitro and in vivo.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3234 – A0269.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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