The Effects Of Hypothermia On Autophagy After Traumatic Brain Injury And Study Of Its Mechanisms

Traumatic brain injury(TBI) causes high mortality and morbidity, the pathological basis of which include primary insult resulting from direct mechanical strike, delayed brain damage and cell death, including apoptosis and autophagic cell death caused by secondary insult. After studies on apoptosis after TBI in the past decades, we have get clear understanding of its development and signal pathways. However, function of autophagy in TBI is still controversial. Many experimental and clinical studies have revealed the neuroprotective effect of hypothermia after TBI, which could attenuate the pathological insults of brain tissue after TBI, reduce cell apoptosis and improve recovery of neural function. However, its neuroprotective mechanisms, especially effect of autophagy in the process are still unclear. Present study adopted fluid percussion TBI model(normothermia and hypothermia) to observe the changes of apoptosis and autophagy and explore the neuroprotective effect of hypothermia on TBI through the point of autophagy.This study includes three parts. Part one: establishment and evaluation of hypothermia fluid percussion TBI models in rats; Part two: effect of hypothermia on the apoptosis and autophagy in cortex and CA1 region of hippocampus after fluid percussion TBI; Part three: modulation of autophagy on the motor and cognitive functions after fluid percussion TBI.Part one: Establishment and evaluation of hypothermia fluid percussion TBI models in ratsObjective: To establish stable and reusable normothermia and hypothermia fluid percussion TBI model in rats and evaluate the animal model from two aspects: histopathological analysis and neurobehavioral assessments. On basis of the model, in-depth experiments were carried out.Methods: 72 adult male SD rats were randomly assigned into four groups: sham injury with normothermia group(n=18), sham injury with hypothermia(n=18), TBI with normothermia(n=18) and TBI with hypothermia(n=18). TBI model was induced with fluid percussion TBI device and moderate hypothermia(32.5±0.5℃) was achieved by partial immersion in a water bath(0℃) under general anesthesia for 4 hours. Then the brain temperature was adjusted to 37±0.3℃ slowly in one hour. Rats in sham injury with normothermia group underwent all surgical and anesthetic procedures except TBI and hypothermia treatment; rats in sham injury with hypothermia group underwent all surgical and anesthetic procedure and hypothermia treatment except TBI; rats in TBI with normothermia group underwent TBI procedure except hypothermia treatment; rats in TBI with hypothermia group underwent TBI procedure and hypothermia treatment. Physiological parameters and reflexes changes of all rats were monitored. Rats were sacrificed at 6h or 24 h after TBI. Gross appearance of the brain and histopathological changes with H&E staining were performed in 3 rats of every group at 6h and 24h after TBI. 6 rats in every group was used for motor test with beam walk test and Morris water maze test.Results: All physiological parameters were within normal ranges for all four groups, with exception of brain temperature and rectal temperature. Gross appearance of brain demonstrated no obvious abnormal cortical changes in sham injury within both normothermia and hypothermia groups while in TBI with normothermia and hypothermia groups, there were obvious changes, including subdural hematoma, subarachnoid hemorrhage and cerebral contusion and laceration, however, the extent of injury was relatively slight. H&E staining showed a great improvement of brain injury in the cortex and CA1 region of hippocampus in the hypothermia treatment group than that of the normothermia group. Neurobehavioral tests analysis indicated post-TBI hypothermia treatment could significantly improve the motor and cognitive function associated with TBI.Conclusion: We have successfully established a normothermia and hypothermia treatment model after moderate fluid percussion TBI in rats, which paved way for the next stage of our studies.Part two: Effect of hypothermia on the apoptosis and autophagy in cortex and CA1 region of hippocampus after fluid percussion TBIObjective: To explore the changes of apoptosis and autophagy after moderate fluid percussion TBI and post-TBI hypothermia in hippocampal CA1 region and cortex.Methods: 108 rats were randomly assigned to three groups: sham injury with normothermia(n=36), TBI with normothermia(n=36) and TBI with hypothermia(n=36). Rats were sacrificed at 6h or 24 h after TBI. TUNEL staining was performed to analyze the cell death index in cortex and hippocampal CA1region; immunofluorescence technique and Westernblot were used to analyze the changes of apoptosis-related protein caspase-3 and autophagy-related protein, including LC-3 and Beclin-1; double immunofluorescence techniques were also adopted to localize the expression of autophagy in the ipsilateral cortex; transmission electron microscope was used to observe the changes of autophagosomes in ipsilateral cortex and hippocampal CA1 region.Results: There were no significant changes of apoptosis and autophagy in the ipsilateral cortex and hippocampal CA1 region in rats of sham injury with normothermia group. TUNEL staining showed that fluid percussion TBI could induce upregulation of apoptosis index in ipsilateral cortex and hippocampal CA1 region while post-TBI hypothermia treatment could significantly inhibit apoptosis. Caspase-3 protein levels in the TBI normothermia group were significantly higher than that in the TBI hypothermia group. Autophagy-related proteins levels, including Beclin-1 and LC3 were both up-regulated in the TBI normothermia group and further up-regulated in the TBI hypothermia group. Results of TEM demonstrated that number of autophagosomes was increased after TBI and further increased after post-TBI hypothermia. Autophagy was expressed in both neurons and glias.Conclusion: Moderate TBI could induce apoptosis and autophagy in ipsilateral cortex while post-TBI hypothermia could decrease the apoptosis and increase the expression of autophagy.Part three: Modulation of autophagy on the motor and cognitive functions of rats after fluid percussion TBIObjective: To investigate the effect of administration of 3-MA to the neuroprotective effect of post-TBI hypothermia, including apoptosis, autophagy and neurobehavioral outcomes.Methods: 63 rats were randomly assigned into three groups: TBI with hypothermia, TBI with hypothermia and saline and TBI with hypothermia and 3-MA. TUNEL staining was performed to analyze the cell death index in cortex and hippocampal CA1 region; immunofluorescence technique and Western-blot were used to analyze the changes of apoptosis-related protein, including caspase-3, Bcl-2 and Bax; changes of autophagy related protein, including cathepsin, Beclin-1 and LC-3. Transmission electron microscope was used to observe the changes of autophagosomes in ipsilateral cortex and hippocampal CA1 region. 6 rats in every group was used for motor test with beam walk test and Morris water maze test.Results: Protein expression levels of LC3 and Beclin-1 was significantly decreased while that of caspase 3, Bcl-2 and Bax was up-regulated after pre-TBI administration of 3-MA; ratio of Beclin-1/Bcl-2 and Bcl-2/Bax were significantly decreased; TEM demonstrated decrease of number of autophagosomes; Morris water maze test and beam walk test demonstrated deterioration of the neurobehavioral test; Pre-TBI administration of 3-MA could increase the cell death index.Conclusion: Upregulation of autophagy may be one possible mechanism of the neuroprotective effect of post-TBI hypothermia and the possible mechanism may be negative modulation of apoptosis.