Abstract
Purpose: :
To analyze the effects of two novel topical analgesics on corneal epithelial cell migration, toxicity, and wound healing.
Methods: :
Single cell migration studies were performed on primary corneal epithelial cells plated onto glass bottom dishes and incubated in various concentrations of neurotensin receptor agonists (SAR001 and SAR002), proparacaine, or tetracaine. Time-lapse imaging was used to measure the change in two-dimensional position of individual cells over time. Subsequently, the effects of the drugs on wound closure were analyzed by imaging the closure of scratches created within confluent monolayers of cells, under the same conditions used for the motility assay. In addition, cytotoxicity was assessed by treating confluent monolayers with the compounds for 45 minutes, and then adding both ethidium homodimer-1 and calcein-AM to identify dead and live cells, respectively. Finally, the in vivo effects of SAR001 and SAR002 were examined by using fluorescence imaging to monitor the closure of a 6.5mm diameter epithelial defect in rabbit corneas. The rabbits received either 1mg/mL of a neurotensin receptor agonist or 0.5 % tetracaine topically every 2 hours.
Results: :
Cells treated with SAR001 and SAR002 demonstrate velocities comparable to those of untreated cells at all doses, while cells treated with proparacaine and tetracaine exhibit marked decreases in a dose dependent manner. In addition, epithelial cell monolayers treated with all doses of SAR001 and SAR002 exhibit 90-100% wound closure within 24 hours. However, monolayers treated with proparacaine or tetracaine show significant delays in scratch closure, with complete inhibition at concentrations above 1.0 mM. Furthermore, cells treated with SAR001 and SAR002 experience minimal toxicity at all concentrations, relative to control. In contrast, there is a sharp decline in cell viability, with 100% toxicity at concentrations above 5mM, when cells are treated with proparacaine or tetracaine. In vivo studies also show that tetracaine significantly delays epithelial wound healing, whereas wounded rabbits treated with SAR001 and SAR002 show rates of healing comparable to those seen in untreated controls.
Conclusions: :
SAR001 and SAR002 induce minimal toxicity, do not inhibit cell motility, and have minimal effects on corneal epithelial wound healing, both in vivo and in vitro. These findings suggest that neurotensin receptor agonists may be attractive options for use as topical analgesic agents.
Keywords: cornea: epithelium • drug toxicity/drug effects • wound healing