Efficacy of Neuropeptide Alpha-Melanocyte Stimulating Hormone (α-MSH) on Corneal Endothelial Cell Regeneration following Injury

Abstract

Purpose : Corneal Endothelial Cells (CEnC) maintain normal corneal hydration. Damage to the corneal endothelium can lead to persistent corneal edema due to limited CEnC proliferative capacity. Studies have shown a concomitant decrease in density and/or function of both corneal nerves and CEnC following ocular injuries, suggesting nerves exert trophic effects on CEnC. Here we investigate the efficacy of neuropeptide α-MSH on CEnC proliferation and function following injury.

Methods : Wound healing was evaluated using CEnC derived from a healthy 21-year-old male donor cultured in Chen’s medium supplemented with/without α-MSH (10-5M, 10-7M, or 10-9M) for 48hrs. Corneal injury was induced by transcorneal freezing in BALB/c mice (n=8/group) by placing a -72^oC 2 mm-diameter steel rod on the center of the cornea. Mice received 10µL subconjunctival injections of α-MSH (10-4M) or PBS (control) twice weekly for two weeks. Mice were injected with 5-ethynyl-2’-deoxyuridine (EdU) every 6 hours for 48hrs, and CEnC proliferation was evaluated by frequency of EdU+ cells. CEnC functional differentiation was assessed by comparing mRNA and protein expression of Na+/K+-ATPase by RT-PCR and Western blot, and by measuring central corneal thickness (CCT) via optical coherence tomography (OCT).

Results : Supplementing culture media with 10-5M α-MSH resulted in the fastest wound healing compared to the culture medium alone (P=0.03). In vivo, α-MSH treatment following injury increased the frequency of EdU+-CEnC within the wound area (61.6%±8.3% vs. control: 33.7%±5.0%; P=0.02) after 48hrs. CEnC Na+/K+-ATPase expression decreased 0.35-fold (P=0.01) at the mRNA level and dropped 0.63-fold (P=0.002) at the protein level in the control group 2 wks after the injury relative to values before injury; there was also a significant edema at 28 days post-injury (CCT:78.1±28.5mm; P=0.04). α-MSH administration restored CEnC Na+/K+-ATPase expression to baseline control values by causing a 2.3-fold increase in mRNA (P=0.04) and a 1.85-fold increase in protein expression (P<0.001) vs. control. α-MSH treatment led to a 54.9±15.0mm decrease in CCT (P=0.03) compared to the control group by 42d post-injury.

Conclusions : Our results demonstrate that following corneal endothelial injury, treatment with α-MSH prevents corneal edema by promoting proliferation of CEnC and maintaining function of mobilized/newly-generated CEnC.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.