Pressure-Related Effects on Homeostatic Müller Cell Proteins in the Adult Porcine in Vitro Retina.

PURPOSE: To explore early pressure-related effects on Müller cell homeostatic proteins in the in vitro adult porcine retina. METHODS: Retinal explants were subjected to 0-, 10-, 30-, or 60-mmHg of pressure for 24 or 48 h in culture. Retinal explants fixed immediately after enucleation were used as controls. Müller cell proteins were evaluated by GFAP, GS, CRALBP, and bFGF immunohistochemistry. RESULTS: GFAP-labeling revealed no differences in fluorescence intensity after 24 or 48 h in any of the pressure groups compared with control retinas. However, a higher intensity was found in the 30- and 60-mmHg groups compared with 0-mmHg counterparts after 24 and 48 h. A higher intensity in GS-labeled sections was found in the 10-and 60-mmHg groups compared with controls and remaining pressure groups after 48 h. Compared with control retinas, CRALBP labeling revealed a higher intensity in the 60-mmHg group after 24 h and in the 10-, 30-, and 60-mmHg groups after 48 h. After 24 and 48 h, a lower intensity was found in bFGF-labeled cells in the 0-, 10-, and 30-mmHg groups compared with controls, while no difference was seen for the 60-mmHg group. CONCLUSIONS: Müller cells in the cultured porcine adult retina respond early to pressure by altering the expression of GFAP as well as the homeostatic proteins GS, CRALBP, and bFGF.

Beneficial and Detrimental Pressure-Related Effects on Inner Neurons in the Adult Porcine In Vitro 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.

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 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.