A NEW MODEL FOR RAPID STRETCH-INDUCED INJURY OF CELLS IN CULTURE - CHARACTERIZATION OF THE MODEL USING ASTROCYTES

The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequenc es of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to co nfluency in commercially available 25-mm-diameter tissue culture wells with a 2-mm-thick flexible silastic bottom. A cell injury controller was used to produce a closed system and exert a rapid positive pressur e of known amplitude (psi) and duration (msec). The deformation of the membrane, and thus the stretch of the cells growing on the membrane, was proportional to the amplitude and duration of the air pressure pul se. Extent of cell injury was qualitatively assessed by light and elec tron microscopy and quantitatively assessed by nuclear uptake of the f luorescent dye propidium iodide, which is excluded from cells with int act membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to t he extent of the silastic membrane deformation. Increasing cell stretc h caused mitochondrial swelling and vacuolization as well as disruptio n of glial filaments. Stretching also caused increased dye uptake, wit h maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) c aused little dye uptake. With increasing postinjury survival fewer cel ls took up dye, implying cell repair. LDH release was also proportiona l to the amplitude of cell stretch, with maximum release occurring wit hin 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. O ur continuing experiments suggest that this model can be used to study the biochemistry and physiology of injury as well as serve as a tool to examine the efficacy of therapeutic agents.