The Contribution Of Autophagy Pathway To Neuronal Cell Death And Neuronal Dysfunctions In Mice After Traumatic Brain Injury

Objective: The present study was sought to study the contribution of autophagy/lysosomal pathway to neuronal cell death and and neuronal dysfunctions after traumatic brain injury, and to explore its relevant mechanisms.Methods: Traumatic brain injury (TBI) model in mice was established quantitatively. Mice were pretreated with lateral cerebral ventricle infusion of two autophagic inhibitor 3-Methyladenine (3-MA) and Brefeldin A (BFA). TBI-induced cortical and hippocampal neuronal cell death was evaluated by intraperitoneal injection of propidium iodide. Lesion volume was estimated by campeachy dyeing and stereology microscopic observation, and a subset of mice were studied behaviorally (Motor test and Morris water maze) in our study to detect motor and cognitive function of the injured animals post-TBI. By Immunofluorescence, stereology microscopic observation and western blot analysis, changes of protein expression in autophagy/lysosomal pathway and apoptosis signal pathway were detected: Cathepsin-B, LC3, Beclin-1, Caspase-3, Bcl-2, and Bax in injuried cortex and hippocampus after TBI, and try to study the possible contribution of autophagy/lysosomal pathway to neuronal cell death and and neuronal dysfunctions after TBI.Results: (1) PI positive cell counts: PI-positive cells were found starting from 1 h and 6 h after TBI and increased rapidly at 12 h, peaked at 24 h, but decreased from 48 h. The number of PI positive cells in 3-MA or BFA group was signifcantly different from that in saline group at 24 h respectively (P<0.05). (2) Lesion Volume Measurements: the lesion volume was obviously reduced in 3-MA or BFA treated groups compared with the saline group (P<0.05). (3) Praxiology Experiment: 3-MA and BFA could significantly attenuated motor dysfunctions respectively in 24 h post-TBI (P<0.05), but it was not obviously different from that at later time stages; 3-MA could significantly attenuated learning and memory dysfunctions from 7 d to 10 d post-surgery campared with that in saline group (P<0.05). (4) Protein Expression: in 3-MA group, the mean ratios of the band intensities of cathepsin-B, caspase-3, beclin-1/bcl-2 significantly decreased (P<0.05) and a significant recovery of Bcl-2 /Bax (P<0.05) was observed at 24 h and 48h. Pretreatment with BFA resulted in a significant decrease of LC3-II/LC3-I levels from 24 h to 48 h (P<0.05).Conclusions:1. Autophagy/lysosomal pathway involved in the physiopathologic course of post-TBI. Blockage of autophagy/lysosomal pathway attenuated TBI-induced Neuronal cell death, decreased lesion volume, and improved motor and cognitive functions.2. Autophagy/lysosomal pathway regulated the TBI-induced neuronal cell death and neuronal dysfunction by influencing apoptosis signaling pathway.