The Effects Of Autophagy/Apoptosis/Plasma Membrane Repair After Traumatic Brain Injury And Preliminary Study Of Mutual Regulation Mechanisms

Brain injury is the most important position in forensic violent deaths. Moreover, traumatic brain injury (TBI) is the most important injury.In modern society, the incidence and mortality of TBI in the young aged population, and is one of the major reasons for hospital admissions in their daily lives. Brain damahe related research is not only the important research area in clinical medicine and neurobiology, but also an important part of forensic research. The field of forensic pathology in the past focused on the adverse consequences of brain damage and the action mechanisms of external force. Therefore, it is of great significance for medicine and forensic science to study the mechanisms of cell damage after TBI. Mechanical disruption of neurons triggers a cascade of events leading to mitochondrial and lysosomal membrane permeability damage, tissue edema, and neuronal cell death, even impaired motor and cognitive functions after TBI. Mang studies have addressed TBI-induces cell death mechanisms. Recently, several studies have shown that cell autophagy is increased after TBI.Autophagy is pathway that involved in the dynamic morphological changes of sub-membrane structure, and lysosomal-mediated degradation of proterins and organells. Four aspects are included:the substrate-induced formation of the autophagic precursor (proautophagosome PAS), formation of autophagosome, fusion of autophagosomes with lysosomes, and degradation of autophagosome contents. A breakthough has been made in the understanding of the role of Bcl-2in controlling autophagy via its interaction with Beclin-1. Autophagy is activated, and can coexist or occur sequentially with apoptosis. The increased LC3immunostaining is found mainly in neurons at24h post-TBI. However, no experimental studies have addressed the role of autophagy in TBI-induced cell death and traumatic damage. Based on the above-mentioned findings, we hypothesize that the autophagic mechanisms may participate in TBI-induced brain damage. To confirm this hypothesis, using the relatively selective autophagy inhibitors, the first section of the present study was designed to define the role and mechanisms of autophagy in TBI-induced cell death and brain damage, and assess the relationship between autophagy and apoptosis. On this basis, in the second part, we study the relationship between the regulatory mechanisms of autophagy activation after traumatic brain injury and apoptosis signaling pathways.Previous data demonstrate that p53and its target genes (PUMA and damage-regulated autophagy modulator (DRAM) involved in apoptosis and autophagy. Over-stimulation of glutamate receptors can induce Nuclear Factor-kappa B (NF-KB)-dependent expression of p53in the in vivo excitotoxic model. And NF-KB/p53pathway participates in excitotoxic neuronal death through both apoptotic and autophagic mechanisms. However, few experimental studies have addressed the role of the NF-KB/p53pathway in a mouse model of TBI. Here, roles of NF-KB/p53pathway in TBI-induced autophagy and apoptosis activation were been studied.The loss in cell membrane integrity, consisting of membrane blebbing and altered permeability, has recently been found to be a major contributor to the development of neuronal damage subsequent to traumatic injury by leading to ionic imbalances and activation of several cellular pathways. Poloxamer188(P188) is a nonionic, amphiphilic co-polymer (MW:～8400), including a central hydrophobic molecule which is flanked on both sides by two hydrophilic chains of polyoxyethylene. Having various clinical applications as a surfactant, P188has been found to be capable of sealing damaged cell membranes. A putative membrane-resealing agent P188may reseal injured (permeable) cells after controlled cortical impact in mice. Using cortical and hippocampal neuron cultures, P188was shown to protect neurons from excitotoxic or oxidative stress-related necrosis and from electroporation by inserting into the membrane and inhibiting membrane peroxidation. However, the neuroprotective mechanisms of P188in TBI are not clear. By building an in vitro primary neuron injury model, the presrnt study was aimed to investigate the neuroprotective mechanisms in subsequent effects of the mechanical trauma, eg changes of mitochondrial and lysosomal membrane after TBI.Part I The effects of autophagy on traumatic brain injury-induced membrane integrity damage, lesion volume and neurologic outcome deficitsBackground:Previous data demonstrate that traumatic brain injury (TBI) activates autophagy, and increases micro tubule-associated protein1light chain3(LC3) immunostaining mainly in neurons. However, the role of autophagy in traumatic brain damage remains elusive. The aim of this part in the present study was to investigate the effects of autophagy on TBI-induced cell death, and neurologic function.Methods:Mice were subjected to TBI using an improved weight-drop model. The autophagy inhibitors3-methyladenine (3-MA) and bafliomycin A1(BFA) were administered with a single i.c.v. injection at10min before TBI. The changes in autophagy after TBI were first detected visually with electron microscopy. We next examined LC3expression by double labelling damaged cells in the brain slice with propidium iodide (PI) and anti-LC3. In addition, we examined the mRNA levels of LC3detected by Real-time PCR, and the protein levels of LC3Ⅱ and P62detected by immunoblotting, which have been found to access autophagy previously. PI labeling was used to identify injured cells, and the numbers of PI-positive cells were counted. To explore whether PI-positive cells could represent TBI-induced cell loss, we assessed the cumulative loss of brain tissue on7days after TBI. To further determine whether the reduction in autophagy with3-MA treatment was associated with improved neurologic outcome, we sought to perform behavior experiments, including Motor Test and Morris Water Maze.Results:①Electron microscopy:compared with the sham group, the main changes in ultrastructure of neurons is the increased number of autophagosomes (APs) and macrophage lysosomes (ALs) from1h to24h post-TBI. And the obvious nuclear fragmentation was observed in chromatin of the cell nucleus at48h after TBI.②Thelevels of LC3:The levels of LC3mRNA and LC3Ⅱ protein were significantly upregulated from1h to48h after TBI. Pretreatment with3-MA significantly reduced the relative abundance of LC3mRNA and LC3Ⅱ protein in the injured cortex at6h and24h post-TBI.③The levels of p62protein:the decreased protein levels of p62after TBI, was detected in the injured cortex at6h and24h post-TBI. We found that3-MA and BFA pretreatment both maintained p62levels, versus the saline-treated mice.④Immunostainning of LC3:TBI resulted in altered staining patterns of LC3in cortical brain regions. At24h after injury, the increased LC3immunoreactivity was seen in PI-positive cells located around the injury epicenter and exhibited a punctate staining pattern in the cell soma. The increased punctate LC3-II dots colocalizing with PI-stained nuclei24h after TBI were partially inhibited by3-MA pretreatment.⑤PI cell count:TBI elicited a significant increase in the number of PI-positive cells from12to72h, and the count peaked in the48h group. Pretreatment with3-MA and BFA resulted in a significant decrease in the amount of PI-positive cells at24h and48h post-TBI, respectively.⑥Lesion volume:TBI caused profound tissue loss of the brain, but a single i.c.v. injection of3-MA and BFA significantly reduced TBI-induced lesion volume, versus the vehicle-treated group, respectively.⑦Motor Test:TBI elicited a significant decline in motor performance on days1-4, which returned to basal levels on days5post-injury. In marked contrast, treatment with3-MA (P<0.05) accelerated the recovery of motor functional outcome on days1-3post-TBI.⑧Morris Water Maze:When there were no differences in motor function between groups, Morris-water maze performance was tested on days11-20. All TBI-treated animals displayed increased latencies in the ability to find the hidden platform (P<0.05), versus the sham group on days11-18. After injury, animals subjected to3-MA pretreatment demonstrated a significant decrease in the latencies, relative to saline vehicle mice on days14-17(P<0.05). On days19and20, there were no differences in latencies to find the visible platform between groups (P>0.05). Finanlly,3-MA-treated mice maintained a daily rate of learning similar to sham-injured mice, whereas vehicle-treated mice showed a reduced daily rate of learning in the Morris-water maze (P<0.05).Conclusion:Cell autophagic activity in brain cortex is increased and apoptosis is activated after TBI. The acitivation of autophagy induced by TBI was inbibited by the autophagy inhibitors (3-MA and BFA), suggesting autophagy is involved in the process of brain damage. Treatment with3-MA partially inhibited the increased punctate LC3-Ⅱ dots colocalizing with PI-stained nuclei24h after TBI, indicating a relationship between autophagy and cell death may exist, and autophagy may be involved in neuronal death caused by TBI. In addition, both3-MA and BFA could reduce TBI-induced cell death and lesion volume. Inhibition of autophagy by3-MA could attenuate behavioral outcome, including motor function and cognitive function. These neuroprotective effects of the autophagic inhibitors may be associated with an inhibition on TBI-induced up-regulation of LC3expression, and on down-regulation of p62protein expression. Taken together, these data imply that the autophagy pathway is involved in the pathophysiologic responses after TBI, and inhibition of this pathway may help attenuate traumatic damage and functional outcome deficits.Part Ⅱ Study on the relationship between regulation mechanisms of autophagic activation and apoptosis signaling pathway after traumatic brain injuryBackground:The results of part Ⅰ indicated the autophagy pathway is involved in the pathophysiologic responses after TBI. Previous data demonstrate that p53and its target genes (PUMA damage-regulated autophagy modulator (DRAM) involved in apoptosis and autophagy. NF-κB/p53pathway participates in excitotoxic neuronal death through both apoptotic and autophagic mechanisms. However, few experimental studies have addressed the role of the NF-κB/p53pathway in a mouse TBI model. The main objective of this part was to assess the roles of NF-κB/p53pathway in autophagy and apoptosis activation induced by TBI.Methods:Mice were subjected to TBI using a weight-drop model. The autophagic inhitor3-MA, the NF-κB inhibitor SN50and the p53inhibitor Pifithrin-alpha (PFT-a) were administered with a single i.c.v. injection at10min before TBI, respectively. The changes of time course in the expression of p53and its target genes PUMA and DRAM after TBI were assessed with Real-time PCR and Western blot analysis, and the effects of3-MA on them were also investigated. To exploit NF-κB-dependent p53/DRAM pathway participating in TBI-induced autophagy and apoptosis, the effects of SN50and PFT-a on TBI-induced increases in the levels of p53, and DRAM were detected, respectively. Furthermore, the neuroprotective effects of SN50and PFT-a after TBI were assessed with internucleosomal DNA fragmentation and lesion volume.Results:TBI induced the increases in the expression of p53, PUMA, and DRAM from1h to24h. However,3-MA reversed TBI-induced increases in the expression of p53, PUMA and DRAM at6h and24h post-TBI (p<0.05). SN50and PFT-a could reverse TBI-induced increases in the expression of p53and DRAM at24h post-TBI (p<0.05). TBI caused internucleosomal DNA fragmentation and profound tissue loss of the brain, but treatment with SN50and PFT-a both significantly reduced internucleosomal DNA fragmentation and lesion volume in animals with TBI, versus the saline-treated group (P<0.05).Conclusion:These results imply that TBI can induce NF-κB-dependent expression of p53. NF-KB/p53pathway participates in TBI-induced cell loss and lesion volume through both apoptotic and autophagic mechanisms.Part III:Study on regulation mechanisms of plasma membrane repair in autophagic/lysosomal pathway and apoptosis regulation mechanism after traumatic brain injuryBackground:Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event, which maybe participate the subsequent neuron damage, including autophagic activation and cell death, eg. mitochondrial and lysosomal membrane permeability (MOMP and LMP) damage. The main objective of this part in the current study was to assess the role of the non-ionic surfactant poloxamer188(P188) in MOMP and LMP in response to a well-defined mechanical insult, and to explore whether the neuroprotective effect of plasma membrane repair is through this new mechanism underlying adjusting the autophagic/lysosomal pathway and cell apoptotic pathway.Methods:Using an in vitro cell shearing device (VCSD), mechanical injury produced an immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188or a cathepsin B inhibitor (CBI) after VCSD10min. The protective effect of P188on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ATm) was measured by JC-1staining, and western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also detected. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes.Results:Our results showed post-injury P188treatment increased cell viability, moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria and cathepsin B release from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and inhibit VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188and CBI treatment decreased the cytosolic accumulation of tBid in supermatant of lysosomes, and the amount of mitochondrial localized tBid.Conclusion:These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and such mechanisms are exploited with pharmacological interventions. P188’s neuroprotection appears to be associated with the connection between cathepsin B and tBid-mediated mitochondrial initiation of cell death.