It’s About Time: Time-Dependent Tissue Damage in the Adult Porcine Retina After Enucleation

The ex vivo large animal retina is extensively used in research ranging from discovery of disease mechanisms to future treatment paradigms. Due to limited standardization when harvesting the tissue, the time after enucleation is often ex- tended for several hours, a factor that so far has not yet been fully characterized. The purpose of this study was to investi- gate the relationship between time after enucleation and retinal tissue damage. Adult, porcine retinal explants were dissected and fixed 90 or 240 min after enucleation. In a sep- arate experiment, explants were cultured for 48 h, following dissection either 90 or 240 min after enucleation. Retinas were analyzed morphologically using hematoxylin and eo- sin for overall tissue damage, TUNEL staining for detection of apoptosis, and RBPMS immunohistochemistry for evalua- tion of ganglion cell survival. In addition, medium from the cultured explants was sampled after 2, 24, and 48 h of culture and assessed for the cell damage marker lactate dehydroge- nase (LDH). Retinas examined 240 min after enucleation dis- played a significant increase in overall tissue damage, in- creased apoptosis, and decreased ganglion cell survivalcompared with 90-min counterparts. In the culture experi- ment, no significant difference in overall tissue damage was found between the 2 groups, however, apoptosis was sig- nificantly increased, and ganglion cell survival decreased in the cultured 240-min group. In addition, a significantly in- creased LDH medium activity was found in the 240-min group compared with the 90-min counterpart at all time points. The adult porcine retina is relatively resistant to tis- sue damage 90 min after enucleation but displays distinct signs of injury after 240 min. The importance of these time points is further highlighted when retinal explants are cul- tured. Our results strongly suggest that time after enucle- ation is a crucial factor that should be considered in experi- ments involving the ex vivo adult porcine retina.