A New Model for Rapid Stretch-Induced Injury of Cells in Culture: Characterization of the Model Using Astrocytes

Abstract

The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequences of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to confluency 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 pressure 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 pulse. Extent of cell injury was qualitatively assessed by light and electron microscopy and quantitatively assessed by nuclear uptake of the fluorescent dye propidium iodide, which is excluded from cells with intact membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to the extent of the silastic membrane deformation. Increasing cell stretch caused mitochondrial swelling and vacuolization as well as disruption of glial filaments. Stretching also caused increased dye uptake, with maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) caused little dye uptake. With increasing postinjury survival fewer cells took up dye, implying cell repair. LDH release was also proportional to the amplitude of cell stretch, with maximum release occurring within 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. Our 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. Key words: cell injury; astrocytes; cell culture; traumatic brain injury