摘要
越来越多的研究表明,急性脑损伤后存在神经细胞的坏死和凋亡,而谷氨酸的兴奋性毒性(Glutamic excitability toxicity)及钙离子(Ca2+)内流被认为是整个过程的“发动者”。最新的研究已证实钙整合素结合蛋白(Calcium-and Integrin-Binding Protein, CIB)可与多种蛋白及金属离子相互作用,参与细胞黏附、细胞凋亡、DNA损伤修复等过程,但关于CIB在脑外伤后神经细胞的凋亡过程中发挥着何种作用则未见报道。亚低温能够减轻创伤性脑损伤后病理损害的程度,促进神经功能恢复,但其具体的保护作用机制仍未完全阐明。本研究对通过建立大鼠中度液压颅脑损伤(Moderate traumatic brain injury,MTBI)模型,并给予亚低温的干预措施,运用Western-blot、Real-time PCR、免疫组化等方法观察亚低温对脑损伤后钙整合素结合蛋白(Calcium-and Integrin-Binding Protein, CIB)及N-甲基-D-天冬氨酸受体1(NMDAR1)表达的影响。本实验证明了CIB1、CIB2与神经细胞的凋亡密切相关,而亚低温的脑保护作用与抑制NMDAR1表达,从而减少Ca2+对CIB1、CIB2的抑制相关。
本实验分为三部分：第一部分：大鼠液压颅脑损伤及亚低温模型的建立；第二部分：颅脑损伤后钙整合素结合蛋白表达变化及神经细胞凋亡的研究；第三部分：亚低温对脑损伤后钙整合素结合蛋白表达及其脑保护机制的研究。
第一部分大鼠液压颅脑外伤后常温及亚低温模型的建立
目的建立稳定的常温(normothermia)和亚低温(mild hypothermia)治疗液压冲击脑损伤(Fluid Percussion Injury,FPI)大鼠动物模型,并观察评估模型的实用性及有效性,从而为深入开展创伤性颅脑损伤的相关动物实验研究奠定基础。
方法1.实验动物及分组：雄性SD大鼠30只,随机分为：假损伤组(Sham,n=10)；液压脑损伤常温组(TBI+N,n=10);液压脑损伤亚低温组(TBI+H,n=10)。2.液压脑损伤动物模型建立及亚低温的实施：亚低温组大鼠TBI形成后立即予以冰浴降温,使脑温在15分钟内下降至33.0±0.5℃,并维持该脑温水平4h,之后在1h内复温至正常水平(37±0.3℃),常温组大鼠只致伤不降温,保持脑温在37+0.3℃,假损伤组大鼠仅做致伤前准备而不予以液压打击。所有实验组动物在损伤前15分钟至损伤后4小时均行各项生理参数检测,每组各4只分别在液压颅脑伤前后完成神经反射时间评分测试,行走实验。另取每组各3只于伤后24h行大体标本观察,3只行组织病理学分析(H&E染色)。
结果所有生理参数除体温外均在正常范围内波动无统计差异,而损伤亚低温组脑温和肛温较常温有明显差异(P<0.01)。神经反射时间结果显示在损伤后常温组反射时间明显增加,而在亚低温组反射时间虽然较假损伤组有所增加但是和常温组相比增加幅度明显减弱(P<0.01)。常温组和亚低温组大鼠TBI后行走试验的逃脱时间较致伤前及假损伤组大鼠显著延长(p<0.01),亚低温组大鼠逃脱时间较常温组大鼠有明显缩短(p<0.01),而组织病理学分析显示亚低温组大鼠致伤灶周围皮层和海马脑损伤程度较常温组大鼠明显减轻。
结论我们成功地建立了符合临床上颅脑损伤患者的神经行为学和组织病理学特点的大鼠常温和亚低温颅脑损伤模型,并对其有效性及实用性进行了评估,为下一步亚低温对创伤行脑损伤后细胞凋亡作用机制的研究提供了良好条件。
第二部分颅脑外伤后钙整合素结合蛋白表达变化及神经细胞凋亡的研究
目的检测颅脑损伤对钙整合素结合蛋白表达的影响,并观察颅脑损伤后神经细胞凋亡变化,探讨钙整合素结合蛋白与神经细胞凋亡的关系。
方法第一部分采用侧方液压冲击装置,成功建立了大鼠中度脑损伤模型,在此基础上,将104只SD大鼠随机分为两组：假损伤组(n=13)和损伤组(n=91)。损伤组分又为7个时间点(n=13/时间点)：1h、2h、4h、6h、12h、24h、72h。通过运用实时荧光定量RT-PCR和Western-blot技术来检测大鼠海马CIB1、CIB2在基因和蛋白水平的表达变化,并运用Tunel染色技术来检测神经细胞的凋亡变化。
结果RT-PCR结果显示mRNA的表达损伤组与假手术组相比,CIB1与CIB2在颅脑损伤后显著上调(p<0.01),伤后4h达到高峰,并在伤后72h接近假手术组(p>0.05)。Western-blot结果表明CIB1、CIB2蛋白的表达量在伤后6h达到高峰(p<0.01),在伤后72h与假手术组相比差异无统计学意义(p>0.05)。Tunel染色结果显示颅脑损伤后神经细胞的凋亡在伤后早期(1h)就可见到神经细胞凋亡的增加,伤后6h达到高峰而后逐渐下降至接近假手术组。
结论颅脑损伤后导致海马钙整合素结合蛋白CIB1、CIB2mRNA和蛋白表达的显著改变并呈现出时程的变化；颅脑损伤后钙整合素结合蛋白的表达改变可能与神经细胞的凋亡有密切关系。
第三部分亚低温对颅脑外伤后钙整合素结合蛋白表达及其脑保护机制的研究
目的检测亚低温对创伤性颅脑损伤后海马组织中钙整合素结合蛋白及N-甲基-D-天冬氨酸受体1在基因和蛋白水平的表达变化；初步探讨钙整合素结合蛋白与NMDAR1的关系及亚低温的脑保护作用机制。
方法147只SD大鼠随机分配于假损伤组(n=13)、损伤常温组(37+0.3℃,n=52)和损伤亚低温组(33.0+0.5℃,n=52)。损伤组(常温组和亚低温组)分4h (n=13)、6h (n=13)、12h (n=13)和24h(n=13)四个实验时间段检测。所有损伤组(常温组和亚低温组)每个时间段和假损伤组行实时荧光定量RT-PCR和Western-blot检测(?)CIB1、CIB2、NMDAR1分别在基因和蛋白水平的表达变化(n=5/组)[常温组第二部分已做],并结合Tunel染色方法检测神经细胞凋亡情况(n=3/组)；同时进行神经行为学的观察(n=5/组)。
结果CIB1、CIB2、NMDAR1表达在损伤常温组较假损伤组明显增加(p<0.01),而亚低温组虽较假损伤组增加(p<0.05),但较常温组表达量低(p<0.05)。实时荧光定量RT-PCR和Western-blot结果显示,与假手术组相比,亚低温组钙整合素结合蛋白CIB1、CIB2及NMDAR1在mRNA和蛋白水平的表达显著降低(p<0.05)。Tunel染色显示亚低温组与常温组相比,神经细胞凋亡百分比显著降低(p<0.01)。
结论液压脑损伤能够导致大鼠海马组织中CIB1、CIB2、NMDAR1基因和蛋白表达的上调,并可导致神经细胞凋亡的增加,而亚低温的干预治疗可以减少NMDAR1的表达并能够明显减少神经细胞的凋亡。在亚低温治疗的众多脑保护作用机制中钙整合素结合蛋白发挥着重要的作用,其作用的发挥与NMDAR1的表达相关,揭示了亚低温启动内源性神经保护并从源头上阻断凋亡进程的分子基础及起效机制,从而为脑外伤的治疗开创新的治疗靶点提供理论基础。
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 Injury
Objective 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 Apoptosis
Objective: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 Injury
Objective 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.
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