EEBA Program (PDF/3MB) - EEBA - Annual Meeting

DNA DAMAGE IN DONOR CORNEAL ENDOTHELIUM UPON TRANSFER FROM OPTISOL GS TOORGAN CULTUREK. Ustgård-Andersen¹, K. Haug¹, A. Azqueta², B. Nicolaissen¹, A. R. Collins¹¹Norwegian Cornea Bank, Center for Eye Research, ¹Department of Ophthalmology, Oslo University Hospital, Ullevaaland University of Oslo; Department of Nutrition, Insitute for Basic Medical Sciences, University of Oslo, NorwayPurpose: The upper limit for storage of donor corneas in Optisol GS is relatively short. Transfer of suchtissue to an Eye Bank Organ Culture (EBOC) system may increase the life span, reduce the numberof donor corneas discarded due to expired shelf life, and increase the overall pool of donor tissue.We have recently examined the limbal epithelium and shown that such transfer is compatible with amaintained regenerative potential and expression of key morphological characteristics (Haug K et al.2012). We here examine the endothelium on donor corneo-scleral rims for DNA damage after primarystorage in Optisol GS and after a subsequent incubation for 1 week in Eye Bank Organ Culture (EBOC).Methods: Comet assay (single cell gel electrophoresis) was used to measure DNA damage in cornealendothelial cells before (n=7) and after (n=5) transferring from cold storage to EBOC. Electrophoresisof lysed and enzyme (FPG, Endo III and T4 Endo V)-treated samples results in structures resemblingcomets, observed by fluorescence microscopy. The intensity of the comet tail relative to the headreflects the frequency of DNA altering lesions (Collins AR & Azqueta A 2012).Results: The level of strand breaks was relatively low in both cold-stored tissue and in the tissueincubated in EBOC for 1 week. Samples retrieved after EBOC showed a significant decrease in FPGsensitivesites reflecting less oxidized purines (mainly 8-hydroxy guanine). Accumulation of dimersinduced by UV-light are indicated by the net T4 endo V-sensitive sites, which is observed to be quitehigh in both groups. The amount of oxidized pyrimidines, given by digestion of Endo III is howeversignificantly decreased upon EBOC incubation.Conclusions: We have found that enzyme-sensitive sites relevant for DNA damage in cornealendothelial cells seem to be decreased using a two-step storage procedure. Our findings may beexplained by a detachment of more severely damaged cells, by less infliction of cellular damage,and also by an activation of DNA repair mechanisms in the more physiological micro-environmentafforded by the EBOC system.56