The Effect of Elevated Atmospheric Pressure on the Survival of Retinal Ganglion Cells Using Flexcell Biopress System

Abstract

Abstract: : Purpose: To determine the effect elevated atmospheric pressure on the survival of rat retinal ganglion cells using a unique, specially designed pressure chamber system. Methods: Rat retinal ganglion cells ( RGC ) were cultured using Dulbecco’s minimum essential medium. Cells from the stable culture were transferred into Petri Dish, with cover slips at the bottom separated by a autoclaved sterile piece of tissue paper to allow easy removal of the cover slip. Once filled with the medium, the petri dish were incubated at 37°C for 3 days to ensure proper growth and attachment of the cells to the cover slips. Later the cover slips were transferred into a specialized Flexcell Biopress chamber (Flexcell International Corporation, NC). A pump which delivered air ( 95% air and 5% CO2 ) from the incubator was connected to one of the ports. The other two ports had a pressure gauze and an adjustable valve outlet. Using the Flexcell software a 2 hour compression regimen was designed and implemented to pressurize the cells at 100mm hg for 2 hr. Following the pressure treatment the RGC’s were incubated for another 24 hr period. The control group consisted of RGC’s on cover slips which were not subjected to pressure and placed in a separate chamber . At different time points after pressure initiation coverslips were removed from the incubator and fixed using 4% paraformaldehyde in PBS at pH 7.5 for 10 minutes. Apoptosis detection by TUNEL and Annexin V kits was done at different time-points. Results: Experimental group had more apoptotic cells compared to the control. Early apoptosis was detected by the Annexin kit and the apoptotic cells stained brightly with fluorescent. There were few necrotic cells present. Apoptosis increased further 24-hours post-pressure treatment. Conclusions: Rat ganglion cells undergo apoptosis when subjected to increased pressure, especially during the first 24 hours. Further studies are required to investigate the possible role of anti-apoptotic agents in enhancing the survival of RGC’s.

Keywords: apoptosis/cell death • ganglion cells • intraocular pressure