An In Vitro System for Studying the Effect of Ambient Hydrostatic Pressure on Growth, Morphology, and Biochemical Aspects of Ganglion Cells

Abstract

Abstract: : Purpose: To develop a system that permits direct visualization of the morphologic and growth characteristics of ganglion cell cultures exposed to varying ambient atmospheric pressure. Methods: An incubation pressure chamber was coupled with a Constar temperature videomicrography system. The pressure chamber was constructed by placing a 4.7-cm x 7.7-cm plastic slide on top of a rubber gasket. Another gasket (0.1 nun) was placed onto the slide and covered with a transparent piece of lexan modified to allow inflow and outflow of tissue culture medium. The assembly was secured between two aluminum plates held together with evenly spaced screws. Ganglion cell cultures were placed onto plastic slides immersed in supplemented minimum essential medium. After a monolayer was established with 80% confluence, the slide was secured in the pressure chamber. Cells were exposed to hydrostatic pressure of 0, 15, 30, 40, 50-mm Hg. Cells were monitored continuously by phase-contrast time lapse videomicrography for 24 hours at 37 degree C. Cell survival was measured with Live/Dead viability kit using esterase activity as marker. Results: Cells grown for 24 hours at low pressures (< 1mm Hg) with a 2.5 mcl/min flow rate had a normal morphology and rate of mitotic activity. Cells remained in a monolayer on the substrate of the slide. Cells exposed to 15-50 cm of pressure for 24 hours exhibited increased rate of cell death as well as apoptosis. Apoptosis was confirmed with annexin staining. Degree of apoptosis correlated with hydrostatic pressure (p<0.01) Conclusions: This system allows systematic study of pressure and drugs on ganglion cell cultures. Ambient pressure higher than 15 mm Hg induces apoptosis and leads to ganglion cell death. A constant flow of medium through the chamber provides a means of effluent collection for biochemical analysis.

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