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.
Purpose To explore pressure-related effects in the adult porcine retina in vitro. Methods Retinal explants were subjected to 0, 10, 30, or 60 mmHg of pressure for 24 or 48 hours in culture. Overall tissue damage in sections was assessed by lactate dehydrogenase media levels, hematoxylin and eosin staining, and TUNEL staining. Inner retinal neurons were evaluated by protein kinase C alpha (rod bipolar cells), CHX10 (overall bipolar cell population), parvalbumin (amacrine cells), and RBPMS (ganglion cells) immunohistochemistry. Results All retinas kept in culture displayed increased pyknosis and apoptosis compared with directly fixed controls. The 10-mmHg explants displayed attenuation of overall tissue damage compared with the 0-, 30-, and 60-mmHg counterparts. No difference in the number of rod bipolar cells was seen in the 10-mmHg explants compared with directly fixed controls, whereas significantly fewer cells were detected in the remaining pressure groups. No difference in the number of ganglion cells in the 0-, 10-, and 60-mmHg groups was seen compared with directly fixed controls after 24 hours, whereas a lower number was found in the 30-mmHg counterpart. A decline of ganglion cells was found in the 0-, 10-, and 60-mmHg group after 48 hours, but no further decrease was seen in the 30-mmHg group. No differences were detected in overall bipolar and amacrine cells in the pressure groups after 24 hours compared with directly fixed controls. Conclusions A moderate amount of pressure attenuates culture-related retinal neurodegeneration. Rod bipolar cells are specifically vulnerable to excessive pressure. Translational Relevance These findings are relevant for glaucoma-related research.
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