EVIDENCE SUPPORTING A ROLE FOR PROGRAMMED CELL-DEATH IN FOCAL CEREBRAL-ISCHEMIA IN RATS

Background and Purpose: Cells die by one of two mechanisms, necrosis o r programmed cell death. Necrosis has been implicated in stroke and oc curs when the cytoplasmic membrane is compromised. Programmed cell dea th requires protein synthesis and often involves endonucleolytic cleav age of the cellular DNA. We assessed the potential contribution of pro grammed cell death to ischemia-induced neuronal death. Methods: Cycloh eximide (protein synthesis inhibitor, 1 mg/kg per 24 hours) or vehicle (1 mL/kg per 24 hours) was continuously infused into the right cerebr al ventricle of spontaneously hypertensive rats. Neocortical focal isc hemia was produced by tandem occlusion of the right common carotid art ery and the ipsilateral middle cerebral artery. After 24 hours the bra in was stained with 2% 2,3,5-triphenyltetrazolium and the ischemic zon e quantitated. Protein synthesis was determined by [H-3]methionine inc orporation into acid-precipitated protein. DNA integrity was determine d in isolated DNA by gel electrophoresis and in whole cells by flow cy tometry. Results: Continuous cycloheximide infusion caused approximate ly 70% reduction in cortical protein synthesis. Cycloheximide also red uced the size of the infarction produced by focal cerebral ischemia wh en compared with controls (ischemic brain volume, 147.5+/-25.9 and 188 .7+/-16.8 mm3 for cycloheximide and saline, respectively; P<.01), sugg esting that protein synthesis may contribute to cell death. Purified D NA from the ischemic zone showed evidence of endonucleolytic degradati on when fractionated by gel electrophoresis. Flow cytometric analysis demonstrated increased propidium iodide fluorescence in intact cells i solated from ischemic cortex, indicating an increased accessibility of degraded DNA to the intercalating dye. Conclusions: New protein synth esis appears to contribute to ischemic cell death in which endonucleol ytic DNA degradation is apparent. These observations implicate program med cell death in ischemic injury and may open unique therapeutic appr oaches for the preservation of neurons in stroke. (Stroke.