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

Abstract

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 extended for several hours, a factor that so far has not yet been fully characterized. The purpose of this study was to investigate 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 separate experiment, explants were cultured for 48 h, following dissection either 90 or 240 min after enucleation. Retinas were analyzed morphologically using hematoxylin and eosin for overall tissue damage, TUNEL staining for detection of apoptosis, and RBPMS immunohistochemistry for evaluation 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 dehydrogenase (LDH). Retinas examined 240 min after enucleation displayed a significant increase in overall tissue damage, increased apoptosis, and decreased ganglion cell survival compared with 90-min counterparts. In the culture experiment, no significant difference in overall tissue damage was found between the 2 groups, however, apoptosis was significantly increased, and ganglion cell survival decreased in the cultured 240-min group. In addition, a significantly increased 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 tissue 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 cultured. Our results strongly suggest that time after enucleation is a crucial factor that should be considered in experiments involving the ex vivo adult porcine retina.

Keywords: Apoptosis; Culture; Enucleation; Large animal model; Porcine retina.