The Effect Of Hypothermia On The Expression Of Calcium-and Integrin-Binding Protein After Traumatic Brain Injury

More and more studies have shown that neurons undergo necrosis and apoptosis after traumatic brain injury.Excitotoxicity of glutamic acid and calcium influx is considered to be the "initiators" of the entire process. Most recent studies have confirmed that calcium and Integrin-binding proteins (CIB) interacts with a variety of proteins involved in apoptosis,DNA damage and repair,but it’s still unknown the role it plays in nerve cell apoptosis. It was reported both by experimental and clinical studies that mild hypothermia can reduce the degree of pathological lesion after traumatic brain injury and is conducive to the neural function recovery,but the specific protection mechanisms have not yet fully elucidated.In the present study, we successfully established moderate traumatic brain injury(MTBI) model in rats which were then treated by mild hypothermia. Real time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to examine the expression of CIB1, including its time and space changes. This experiment proved CIB1is closely related to nerve cell apoptosis, and the brain protective effect of mild hypothermia may be associated with lower CIB1expression.Our whole study comprises of three main parts:1) The construction of moderate lateral fluid percussion injury model with hypothermia or normothermia treatment using rats.2) The effect of traumatic brain injury on the expression of calcium-and integrin-binding protein and apoptosis.3)The effects of mild hypothermia on the expression of CIB and apoptosis after traumatic brain injury PART I Establishment of a Normothermia and Mild Hypothermia Rats Models after Fluid Percussion Brain InjuryObjective To establish a stable rat model of mild hypothermia and normothermia after fluid percussion brain injury. The practicality and effectiveness of the model was assessed so as to so as to lay the foundation for future research.Method1. Experimental animals and groups:30male SD rats were randomly divided into three groups:sham-operated group (n=10); fluid-percussion brain injury of normothermia (n=10); fluid-percussion brain injury of mild hypothermia group (n=10).2. Fluid percussion brain injury animal model and implementation of mild Hypothermia:The TBI model was induced by a fluid percussion TBI device. Mild hypothermia (33.0±0.5℃) was achieved by partial immersion in a water bath (0℃) under general anesthesia for4h. And then the brain temperature was adjusted back to37±0.3℃slowly in one hour. The animals in normothermia group after injury were maintained at body temperature (37±0.3℃); The sham group underwent the same surgical preparation but not the injury. Physiological parameters were assessed for each group from15min before injury to4h after TBI or sham procedures.Four rats in every group finished the nerve reflexes score and beam walking task before and after TBI were induced. After24hours,The whole specimens were observed and the histopathological evaluation was performed with H&E staining in three rats of every group.Results All physiologic variables, with the exception of temperature, were within normal ranges for both normothermia group and hypothermia group. In contrast, the brain temperature and rectal temperature of hypothermia rats were significantly decreased, compared with those of the normothermia rats after TBI (p<0.01). The time of nerve reflexes was up-regulated in rats of normothermia group24h after TBI, while post-traumatic mild hypothermia, initiated immediately after the insult and maintained for a period of4h, significantly attenuated time up-regulation following TBI(p<0.05). Both the latency for the rat to reach the goal box of beam walking task in TBI-mild hypothermia and TBI-normothermia group were significantly delayed than that of sham injury group(p<0.01).The latency of TBI-mild hypothermia group was significantly shorter than that of TBI-normothermia group(p<0.01).The results of histopathologic analyses showed a substantial improvement of neuronal injury in the region adjacent to impact site and hippocampus of the hypothermia group than that of the normothermia group.Conclusion We have succeeded in establishing normothermia and mild hypothermia treatment model after FPI in rats, and the practicability and effectiveness of the model was verified by neuroethology and histopathology. PART II:The Effect of Traumatic Brain Injury on the Expression of Calcium-and Integrin-Binding Protein and Neuronal ApoptosisObjective:This part was aimed to detect the expression of CIB and observe the apoptosis of neurons after brain injury,and then to investigate possible relationship between calcium and integrin-binding proteins and neuronal apoptosis.Method:On the basis of a successful moderate brain injury model in rats,104adult male Sprague-Dawley rats were randomly assigned to two groups:TBI with normothermia (37±0.3℃), and sham-injury.The group of sham-injury were then divided into7groups according to the time after TBI:1h、2h、4h、6h、12h、24h、72h. CIB expression was examined by real time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Tunel staining technique was used to detect neuronal apoptosis.Results:Based on the RT-PCR and Western blotting results,CIB mRNA increased at the beginning of TBI,reached the highest level at4h post trauma,and then decreased until72h after TBI.The expression of CIB proteins was significant up-regulated and reached the highest level at6h post TBI(p<0.01).Tunel staining showed that the percentage of neuronal apoptosis increased at the early stage (1h) afterTBI,reached the highest level at6h and then decreased gradually until72h.Conclusion:TBI is accompanied by an increase in the expression of CIB(CIB1、CIB2) which shows a high-low trend. The high expression of CIB may be related to neuronal apoptosis. PART Ⅲ:Effects of Mild Hypothermia on Expression of Calcium-and Integrin-Binding Protein and Neuronal Apoptosis mechanism after Traumatic Brain InjuryObjective To investigate the impact mild hypothermia on the expression CIB1at gene and protein levels and neuronal apoptosis after traumatic brain injury;To seek a relationship between CIB and neuronal apoptosis by these preliminary exploration..Methods147adult male Sprague-Dawley rats were randomly assigned to three groups:TBI with hypothermia treatment (33.0±0.5℃,n=52), TBI with normothermia (37±0.3℃,n=52), and sham injured control(n=13). TBI model was induced by fluid percussion TBI device. Mild hypothermia (33±0.5℃) was achieved by partial immersion in a water bath (0℃) under general anesthesia for4hours. All the rats (TBI+H,TBI+N)were killed at4h,6h,12h and24h after TBI. The mRNA and protein levels of CIB1of each group were measured using RT-PCR and Western blot techniques, respectively. Tunel staining test was also used to detect neuronal apoptosis(n=12). Neurological behavior was assessed1d～5d and11d～15d post TBI.Results:CIB1、CIB2、NMDAR1mRNA and proteins increased remarkly in the normothermic group after TBI compared with the sham group (p<0.01) while post-traumatic hypothermia could significantly attenuate such effect. According to the RT-PCR and western blot analysis, CIB expression significantly decreased (p<0.05) in the TBI+H group. According to Tunel staining results, the apoptosis index decreased in hypothermia TBI group compared with normothermia TBI group(p< 0.01).Conclusion Our data suggests that moderate TBI would significantly upregulate CIB、CIB2、NMDAR1expression at mRNA and prorein levels, while such an effect could be efficiently suppressed by hypothermia treatment. It indicates the effect of hypothermia on modulation of apoptosis after TBI is a multi-functional way,and the function of hypothermia may be closely related to its effect on NMDAR1.Thus, this study provides a theoretical foundation for clinical application of mild hypothermia after TBI.