2022 Annual Meeting and Alumni Reunion
Category : Basic and Translational Research Candidate : Hamid Alemi Poster #: B2
Efficacy of Alpha-Melanocyte Stimulating Hormone ( α-MSH ) in Suppressing Corneal Edema Following Acute Injury
Hamid Alemi , Shudan Wang , Tomas Blanco , Francesca Kahale , Gustavo Ortiz , Neha Deshpande , Thomas H . Dohlman , Ula V . Jurkunas , Jia Yin , Reza Dana
Purpose : Corneal endothelial cells ( CEnC ) have minimal regenerative capacity and insults to the endothelium may lead to persistent corneal edema . Following ocular injury , the concomitant decrease in density and function of both corneal nerves and CEnC suggests that nerves have a trophic role for CEnC . Herein , we investigate the efficacy of the neuropeptide α-MSH in preventing corneal edema following injury and explore the underlying mechanisms .
Methods : Corneal injury was induced by transcorneal freezing in BALB / c mice ( n = 8 / group ) by placing a -72oC 2 mm-diameter steel rod on the center of the cornea for 10 seconds . Mice received 10 µ L subconjunctival injection of α-MSH ( 10-4M ) or PBS ( control ) immediately post-injury and twice weekly for eight weeks . The following measures were tracked clinically : Central Corneal Thickness ( CCT ) by optical coherence tomography , corneal opacity by slit lamp , and CEnC density by in vivo confocal microscopy . Corneal endothelial tissue was evaluated for apoptosis ( TUNEL assay ), proliferation ( 5-ethynyl-2 ’ -deoxyuridine [ EdU ] incorporation ), and functional differentiation ( Na +/ K + -ATPase mRNA and protein expression ). Immortalized human CEnC ( HCEnC-T21 ) were cultured with / without α-MSH ( 10-7M ) for 24hrs and a scratch assay and live microscopy were used to evaluate wound healing , while CEnC proliferation was measured using the PrestoBlue ® assay .
Results : In the control group , transcorneal freezing led to a 0.61-fold increase in CCT ( vs . baseline : 116 ± 6 , P < 0.001 ) and a 0.38-fold reduction in CEnC density ( vs . baseline : 2954 ± 67 , P < 0.001 ) 8wks post-injury . α-MSH restored CCT to baseline values ( 117 ± 12 µ m , P = 0.004 ) and increased CEnC density by 0.2-fold ( P < 0.001 ). Compared to control , α-MSH treatment led to a 0.61-fold reduction in apoptotic CEnC frequency and a 0.4-fold increase in the number of newly generated ( EdU +) CEnC 48hrs post-injury . Supplementing HCEnC culture with α-MSH led to a 0.25-fold increase in wound healing rate and a 0.62-fold increase in CEnC proliferation ( P = 0.03 ). While control-treated mice experienced a 0.3-fold and 0.63-fold decrease in CEnC mRNA and protein expression of Na +/ K + -ATPase ( P = 0.001 and P = 0.004 , respectively ), α-MSH restored CEnC Na +/ K + - ATPase expression with a 2.3-fold increase in mRNA ( P = 0.044 ) and a 1.85-fold increase in protein levels ( P < 0.001 ) compared to the control group .
Conclusions : Our results demonstrate that following injury , treatment with α-MSH prevents corneal edema through its cytoprotective function and additionally promotes the differentiation and proliferation of newlyformed CEnC .