Investigating retinal therapies using ex vivo retinal explants

• ¹ Tampere University of Technology & BioMediTech, Tampere, Department of Electronics and Communications Engineering, Finland
• ² University of Tampere, Department of Ophthalmology, Finland
• ³ University of Alberta, Flow Cytometry Core, Canada
• ⁴ University of Tampere, Department of Ophthalmology, Finland
• ⁵ Tampere University of Technology, BioMediTech, Finland
• ⁶ Tampere University of Technology, Department of Electronics and Communications Engineering, Finland

Motivation Retinal degenerative diseases cause an enormous economic burden to the society and decrease the life quality and independence of affected patients. Currently, there are no therapies that would cure these diseases and restore vision. Thus, new treatments are urgently needed. In vitro retina model systems would facilitate the therapy development, and enable the investigation of the effects, benefits and risks of the medical treatments to the retinal tissue. In this study, we tested an organotypic retinal explant culture system and its potential as a tool for the investigations of retinal therapies, focusing on the effects of drugs to the functioning and morphology of the retina. Material and Methods Retinal tissue was isolated from young C57Bl/6J mice sacrificed with CO2 inhalation and cervical dislocation. Isolated tissue was cut into halves, yielding four retinal explants per animal. Retinae were cultured for several days on culture membranes under ex vivo conditions in serum free medium supplemented with B27 and N2, as described previously [1, 2]. Electrophysiological and immunohistochemical methods were used to follow the effects of retinal drugs on the morphology, viability and functionality of the retinal tissue. As a test drug, we used rapamycin, an immunosuppressant used in transplantation therapies. All procedures were conducted under dim red light. Electrophysiological characterization of the retinae was performed using multielectrode array technique to record neuronal responses of retinal ganglion cells (RGCs). We used perforated multielectrode arrays (60pMEA200/30iR by Multi Channel Systems MCS GmbH) and the retinae were perfused with Ames’ solution buffered with sodium bicarbonate and equilibrated with a gas mixture of 95% O2 and 5% CO2. Both spontaneous and light-evoked activity was recorded from fresh retinae and retinae after drug treatment. As light stimuli, we used full-field flashes and steps of 505 nm light. Signals were acquired by the MC_Rack data software (Multi Channel Systems MCS GmbH) at a sampling rate of 20 kHz and analyzed with NeuroExplorer (Plexon) and MATLAB (MathWorks). Results Rapamycin induced changes both in the functionality and the morphology of the retinal tissue during our 7-day follow-up culturing period. MEA recordings revealed changes in spontaneous electrical activity of RGCs. Rapamycin treatment reduced the amount of active channels by 19 % during the first 2 days in culture and by 98 % during the 7-day follow-up period compared to control retinal explants. The number of detected spikes decreased by 46 % by day 2 and by 100 % by day 7 compared to control retinae. Remarkable rapamycin-induced changes in peak-to-peak amplitudes could not be observed by day 2. However, peak-to-peak amplitudes decreased by 52 % (average) and by 72 % (maximum) by day 7 compared to control retinal explants. Immunohistochemical analysis showed changes in retinal morphology, especially regarding the reactive gliosis. During the culturing of retinal explants, strong reactive gliosis was observed in control retinae. However, the same phenomenon was absent in retinal explants exposed to rapamycin treatment. Thus, rapamycin treatment remarkably prevented gliosis, but at the same time decreased the signaling of RGCs. Discussion Our results demonstarte that organotypic retinal explant culture systems combined with electrophysiological recordings and immunohistochemical analysis are useful in the studies of retinal therapies, especially regarding the effects of pharmaceutical treatments on the retinal neuronal tissue. The observed changes in retinal neuronal activity and retinal morphology revealed new insight on the functioning of our test drug, rapamycin, although further studies are needed for their verification. Overall, the findings of our studies indicate that parallel morphological and physiological studies are essential to ensure full understanding of the drug effects not only on the viability of the retinal tissue but also on its functionality. Conclusion The motivation for this study was to develop and test a culture system for ex vivo retinal explants to investigate the effects of medical treatments on the retinal tissue. We observed changes in the functionality and morphology of retinal explants induced by our test drug. This culture system thus represents a valuable tool in biomedical research for the studies of the responses of retinal tissue to pharmalogical compounds. References [1] Bull N. et al. Invest Ophthalmol Vis Sci. 52:3309-3320, 2011. [2] Johnson T. and Martin K. Invest Ophthalmol Vis Sci. 49:3503-3512, 2008.