Chelsea Brown

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

Oxygen (O₂) uptake by cells and tissues is a critical indicator of metabolic demand, changes in microenvironment, and pathophysiology. O₂ uptake from the atmosphere accounts for virtually all the O₂ consumption in the avascular cornea; however, a detailed spatiotemporal profile of corneal O₂ uptake (COU) remains undetermined. Here, we used a non-invasive self-referencing optical fiber O₂ sensor—the scanning micro-optrode technique (SMOT)—to report the O₂ partial pressure and flux variations at the ocular surface of rodents and non-human primates. In vivo spatial mapping in mice revealed a distinct COU, characterized by a centripetal gradient with a significantly higher O₂ influx at the limbus and conjunctiva regions than at the center of the cornea. This regional COU profile was reproduced ex vivo in freshly enucleated eyes. The centripetal gradient was conserved across the following species analyzed: mice, rats, and rhesus monkeys. In vivo temporal mapping in mice showed a significant increase in the O₂ flux in the limbus in the evening compared to other times. Altogether, the data unveiled a conserved centripetal COU profile, which may be associated with the limbal epithelial stem cells residing at the intersection of the limbus and conjunctiva. These physiological observations will serve as a useful baseline for comparative studies with contact lens wear, ocular disease, diabetes, etc. Moreover, the sensor may be applied to understand the responses of the cornea and other tissues to various insults, drugs, or changes in the environment.