Abstract
Purpose :
Corneal Schwann cells (cSCs) ensheath axons and are believed to play a vital role in the health and regeneration of the cornea, including its sensory functions. Here we have exploited a nitrogen mustard (NM) corneal injury model to examine cSC and axonal degeneration/regeneration post injury and also tested a novel drug micellar formulation RM4404 that targets cSCs as a potential therapeutic.
Methods :
Transgenic mice harboring the proteolipid protein 1 promoter enhanced green fluorescent protein (PLP-eGFP) gene construct that marks cSCs were used. Topical 0.2% NM solution was applied for 5 min under sedation followed by a phosphate buffered saline washing. Over days 8 to 14, mice were treated twice per day with a topical micellar formulation RM4404 while the control group received vehicle Kolliphor HS-15. Corneal mechanosensitivity scoring by esthesiometer was performed. Mice were euthanized and corneal flat-mounts were stained with axonal β3-tubulin antibody. Fluorescently stained tissues were imaged using epifluorescence microscopy. Tiled images of the corneal staining were assembled using Leica software. Axonal and cSC networks were measured using FIJI Neuroanatomy plugin.
Results :
We found that NM injury causes degeneration of cSCs and associated axons which remained significantly reduced during the 14-day post injury period (P=0.0025; P=0.0283). RM4404 treatment supported the regeneration of cSCs at 14 days post injury (dpi), showing significantly greater numbers in drug treated over vehicle-treated groups (P=0.0012), and RM4404 also supported the regeneration of axons at 14 dpi (P=0.0006). Comparisons of central versus peripheral corneal regions revealed that regeneration was greater at the peripheral regions for both cSCs and axons in RM4404 treated corneas compared to vehicle controls (P=0.0002; P=0.0002). Mechanosensory testing revealed sensory reduction at 7 dpi that remained low in vehicle treated mice at 14 dpi (P=0.0015), while RM4404 treated mice at 14 dpi showed significant restoration achieving corneal sensitivity of injured mice (P=0.7627).
Conclusions :
cSCs are vulnerable to DNA targeting agents, such as NM. Surviving cSCs in the corneal periphery can regenerate and promote trophic support for injured axons to regeneration. For the first time, we provide evidence that pharmacological targeting of cSCs can be exploited for axonal regeneration and restoration of corneal sensory function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.