Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea

Abstract

Although sensory reinnervation occurs after injury in the PNS, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify subbasal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of subbasal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7d after superficial trephination, subbasal axon density returns to control levels; by 28d the vortex reforms. Although axon density is similar to control 14d after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14d, axons retract from the center leaving the subbasal axon density reduced by 37.2% and 36.8% at 28d after dulled blade and rotating burr wounding, respectively, compared to control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration associated genes (RAGs) involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7d after injury and by 14d and 28d after wounding, many of these basal cells undergo apoptosis and die. While subbasal axons are restored to their normal density and morphology after superficial trephination, subbasal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14d after corneal debridement may destabilize newly reinnervated subbasal axons and lead to their retraction towards the periphery.

Topical Mitomycin-C enhances subbasal nerve regeneration and reduces erosion frequency in the debridement wounded mouse cornea

“…There are no significant differences between subbasal axon density at the corneal center after MK and 1mm debridement wounds compared to controls. The data in Figures 2 and 3, along with data showing that subbasal axon density is significantly reduced at the corneal center 28 days after 1.5 mm debridement wounds (Pajoohesh-Ganji, et al, 2015), suggest that chronically elevated corneal epithelial cell proliferation contributes to the inability of the subbasal axons to reinnervate the corneal center after debridement wounding.…”

“…The adjacent image tiles were captured with overlap to ensure proper tiling. Sholl analysis was performed using ImageJ as described in Pajoohesh-Ganji, et al (2015).…”

“…When this occurs at a steady state level, the corneal epithelium remains intact, but if clusters of adjacent basal cells leave the basement membrane and detach from subbasal axons simultaneously, the risk of developing erosions and focal sites of denervation will be increased. A recent study using a mouse model for recurrent erosions showed that 7 days after debridement wounds, clusters of corneal epithelial cells at the corneal center stop proliferating and begin to undergo apoptosis before erosions form (Pajoohesh-Ganji, et al, 2015). The studies presented here ask whether excess corneal epithelial cell proliferation leads to subbasal nerve denervation and erosion formation.…”

Reduced intraepithelial corneal nerve density and sensitivity accompany desiccating stress and aging in C57BL/6 mice

“…Ntn1 and Unc5B are expressed by corneal epithelial, and stromal cells (Han et al, 2012). In response to corneal debridement injuries that lead to development of recurrent erosions, expression of Ntn1, Unc5b, DCC, Efna4, Efna5, and Rgma mRNAs within corneal epithelial cells change over time as erosions form and ICN retraction is observed (Pajoohesh-Ganji et al, 2015). …”

“…All images were acquired using the same intensity settings. Sholl analysis was performed using ImageJ as described previously (Pajoohesh-Ganji et al, 2015). …”