Abstract
The cornea is the transparent layer that covers the front of the eye. Its primary roles are to refract light to the retina and serve as a protective barrier from physical and chemical insults. For the cornea to maintain proper function, the epithelium must sustain its integrity through cell proliferation and healing. To do this, continuous culling and renewing of corneal epithelial cells are essential; therefore, proper migratory cues coupled with healthy stem cell populations are paramount. To maintain this balance of corneal homeostasis and wound healing there must be a healthy population of resident stem cells called limbal epithelial stem cells (LESCs). LESCs are found in the limbus which is where the cornea and conjunctiva intersect. Once activated they become highly proliferative and can undergo symmetric and asymmetric divisions to maintain the population and generate transit amplifying cells (TACs) and other progenitors. During times of wound healing, LESCs proliferate at higher rates and TACs move to seal the wound edge more quickly. Since where and when these cells move to close wounds is one of the most important elements of wound healing, it is essential to understand the signals that guide them.
In recent years, electric signaling has become apparent as a powerful signal of epithelial wound healing. In vitro, different cell types respond to weak direct current (DC) electric fields (EFs) galvanotropically (change in cellular polarity with relation to the EF) and subsequently galvanotaxically (migration of the cell directionally in the EF). Electric signaling has also been shown to affect and be affected by intracellular signaling pathways such as PI3K, AKT, and cGMP. In vitro studies have also shown that electric stimulation regimes can differentiate stem cells into terminal subpopulations.
There is evidence that electrical signaling is an active response to injury rather than a result of cellular leakage and therefore likely modulates cellular migration and proliferation. The cornea naturally has a bioelectric signature during times of homeostasis—a trans-epithelial potential that has been measured up to 45mV. Wounding the cornea results in the generation of various ion fluxes—namely, Cl-, NA+, K+, and Ca2+—that add together to create a heightened endogenous electric current. Moreover, these individual ion fluxes have distinct time course signatures, dissipating at different rates.
Although it is apparent these ion fluxes modulate wound healing, the underlying mechanisms are not yet understood. We hypothesized that ion channels and pumps serve as the “molecular generators'' of the wound current during times of healing and that their transcription would significantly increase. We also hypothesized that stem cell populations are differentially affected by ion fluxes based on their proximity to these “molecular generators”; thus, we expected to find spatially dependent transcription profiles near stem cell populations. To explore these hypotheses, we performed a spatial transcriptomic experiment on mouse cornea at 1 hour, 6 hours, and 12 hours post wounding as well as on an unwounded control.
Surprisingly, we found extremely low levels of detectable stem cell transcripts in the cornea and only K+ (KCNJ14) and cyclic nucleotide-gated channels (CNBG1) were upregulated one hour post wounding. Interestingly, rhodopsin (RHO) and cGMP pathways were significantly enriched one-hour post wounding, along with other components of the phototransduction pathway found in the retina. Further investigation will need to be performed, but these data suggest that known components of the phototransduction pathway play a role in early cornea wound healing in mice.