Comparison of labeling methods and time course of traumatic brain injury-induced cell death in mice

摘要

BACKGROUND:Various molecular mechanisms of cell death following traumatic brain injury have been previously described.However,the time course of cell death remains unclear.TUNEL and Fluoro-Jade B labeling have been widely used to label apoptotic cells and neuronal degeneration.Propidium iodide (PI) functions as a biomarker of cell death in vivo.OBJECTIVE:To explore the role of PI labeling compared to TUNEL and Fluoro-Jade B staining for detecting neural cell death,and to observe time course of traumatic brain injury-induced cell death in mice.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Laboratory of Aging and Nervous Diseases,Soochow University from September 2007 to December 2008.MATERIALS:PI (B1221) was purchased from Sigma,USA.TUNEL kit was purchased from Roche Molecular Biochemicals,USA.Fluoro-Jade B was purchased from Chemicon,USA.METHODS:A total of 70 healthy,male,Kunming mice were randomly assigned to sham-surgery (n=5) and model (n=65) groups.Traumatic brain injury was established using the controlled cortical impact method.PI was intraperitoneally injected at 1 hour prior to animal sacrifice.MAIN OUTCOME MEASURES:TUNEL,Fluoro-Jade B,and PI-positive cells were quantified using a double-labeling method to determine the time course of traumatic brain injury-induced cell death.RESULTS:PI labeled cells in an earlier phase of cell death than TUNEL and Fluoro-Jade B labeling.PI-positive cells were observed immediately following injury,and the numbers rapidly increased in injured brain areas at 1 hour,peaked at 24-48 hours,and subsequently decreased at 3-21 days post-injury.TUNEL-labeled cells were significantly increased at 12 hours,while Fluoro-Jade B-labeled cells were increased at 6 hours after injury,with cells still visible at 6-48 hours post-injury.Moreover,a greater number of PI-positive cells were observed compared to TUNEL-and Fluoro-Jade B-labeled cells.CONCLUSION:PI labeling is more sensitive and reliable than TUNEL and Fluoro-Jade B staining for detecting cell death following traumatic brain injury.Moreover,PI labeling can function as a reliable marker to estimate the entire time course of cell death.