大鼠颅脑创伤后脑红蛋白表达变化的实验研究

目的研究弥漫性颅脑创伤后大鼠脑组织中脑红蛋白的核酸与蛋白表达变化情况，初探脑红蛋白与颅脑创伤的关系。方法选择Marmarou’s自由落体打击装置复制弥漫性颅脑创伤模型，采用实时定量PCR及Westernblotting分别检测伤后不同时间（30min、1h、2h、6h、12h、24h、48h、72h、5d）脑组织中脑红蛋白核酸及蛋白水平的表达情况，并对所得数据进行统计学分析。结果在伤后30min，脑组织中脑红蛋白的核酸表达m现首个高峰，相应地其蛋白表达于伤后1h增高，于伤后2h达峰值；伤后12h，脑红蛋白的核酸表达再次升高，于伤后48h达高峰，其蛋白表达亦于伤后24h增高，至72h达峰值。结论颅脑创伤后脑组织中脑红蛋白呈“双峰”样表达。提示脑红蛋白可拮抗对神经元的创伤应激及伤后继发缺血、缺氧性损害，对创伤后脑组织具有一定的保护作用。     

大鼠弥漫性颅脑损伤后脑红蛋白mRNA变化的实验研究

目的研究弥漫性颅脑损伤后大鼠脑组织中脑红蛋白（NGB）mRNA的表达变化情况，初步探讨NGB与颅脑损伤的关系。方法选择Marmarou自由落体打击装置制作弥漫性颅脑损伤模型．采用实时、定量PCR检测伤后不同时间脑组织中NGB mRNA的表达情况，并对所得数据进行统计学分析。结果在伤后30min，脑组织中NGB mRNA的表达出现首个高峰．此后逐渐下降，至6h恢复至正常水平；伤后12h再次升高，于伤后48h达高峰，此后下降，至伤后5d仍高于正常水平。结论弥漫性颅脑损伤后，脑组织中NGB mRNA表达呈“双峰”，提示其可能参与神经元损伤后应激及继发缺血、缺氧性脑损害的应答机制。     

bFGF、VEGF在大鼠创伤脑组织中的表达

目的研究碱性成纤维细胞生长因子(bFGF)和血管内皮生长因子(VEGF)在大鼠创伤脑组织中的表达及它们之间的关系，从分子水平探讨颅脑损伤后的病理机制，为临床治疗脑损伤的新途径提供实验基础。方法 改进Marmarou大鼠加速弥漫性脑损伤模型，制作成弥漫性轴索损伤同时合并局灶性脑挫伤的新的动物模型，取挫伤灶周围脑组织免疫组化染色观察bFGF、VEGF基因表达情况。结果 脑挫伤灶周围脑组织bFGF基因表达在伤后1h明显增加，伤后12h达高峰；VEGF基因表达伤后逐渐增加，24h达高峰。结论 bFGF、VEGF基因表达与脑损伤密切相关，bFGF、VEGF作为生长因子可能参与颅脑损伤后神经元保护及损伤后修复过程。     

bFGF、VEGF在大鼠创伤脑组织中的表达及关系

目的 研究碱性成纤维细胞生长因子(bFGF)、血管内皮细胞生长因子(VEGF)在大鼠创伤脑组织中不同时间的表达及其它们之间的关系，从分子水平探讨颅脑损伤后的病理机制，为临床治疗脑损伤的新途径提供实验基础。方法 改进Marmarou大鼠加速弥漫性脑损伤模型，制作成弥漫性轴索损伤同时合并局灶性脑挫伤的新的动物模型。SD雄性大鼠48只，随机分为8组。每组6只。取挫伤灶周围脑组织免疫组化染色观察bFGF、VEGF基因表达情况。结果 脑挫伤灶周围脑组织，bFGF基因表达在伤后1h明显增加。伤后12h达高峰；VEGF基因表达伤后逐渐增加，24h达高峰，12h恢复到对照水平。结论 bFGF、VEGF基因表达与脑损伤密切相关，作为生长因子，bFGF、VEGF可能参与颅脑损伤后神经元保护及损伤后修复过程。     

神经生长因子在大鼠脑损伤后血脑屏障的通透性

目的研究神经生长因子（NGF）在大鼠创伤性脑损伤后血脑屏障（BBB）中的通透性，寻找NGF通过BBB进入受伤脑组织的时间窗。方法采用液压打击造成大鼠中、重度颅脑创伤，尾静脉注射伊文蓝（EB），分别于伤后1h，3h，6h，12h，24h，72h，168h测量伤侧脑组织中的EB含量。将纯化的NGF用^125I标记，在上述时间段分别从尾静脉注射碘化NGF，用γ-counter计数仪测量伤侧脑组织的放射活性。结果重度脑创伤后EB值在伤后1h立即快速上升，并在3h达到高峰，峰值持续到72h，随后缓慢下降，在168h EB值仍较对照组明显增高。中度脑创伤后脑组织中的EB的含量在1h开始缓慢升高，6h达到高峰，峰值持续到12h随后下降，在168h基本恢复正常。重度脑创伤后脑组织中^125I—NGF在1h明显升高，在3h达到高峰，峰值持续约24h，在72h明显下降，在168h时仍比对照组高。中度脑创伤后^125I—NGF在1h脑组织中即能检测到，并缓慢升高，在6h即达到高峰，随后下降，至168h时其剂量同正常对照组相仿。结论脑创伤后BBB开放，其开放程度与颅脑创伤程度呈正相关。在脑创伤后NGF能随着BBB的开放而进入脑组织，NGF进入脑组织的量与BBB的开放程度及脑损伤程度呈正相关。     

Fos蛋白和Jun蛋白在犬颅脑枪弹伤局部脑组织的表达

目的　研究犬颅脑枪弹伤后脑神经元早期快反应基因c fos和c jun表达产物Fos蛋白和Jun蛋白的变化规律。方法　 2 0只杂种犬 ,随机分为正常对照组、损伤组。以德国小口径步枪子弹致犬颅脑贯通伤 (PCI)模型为对象 ,采用免疫组化法检测脑组织伤后 30min、2h、6h弹道挫伤区、震荡区及脑干神经元中Fos和Jun蛋白的表达。结果　对照组脑皮质神经元中Fos和Jun蛋白弱表达 ,弹道挫伤区、震荡区及脑干神经元中Fos和Jun蛋白表达于伤后 30min开始增加 ,2h达到高峰 ,6h逐渐下降。且Fos和Jun蛋白表达在弹道震荡区较挫伤区更为明显 (P <0 .0 5 )。结论　Fos蛋白和Jun蛋白在弹道挫伤区、震荡区及脑干神经元均有表达 ,c jun在脑组织内表达的分布范围及变化趋势与c fos基本一致 ,其表达是对损伤刺激的早期反应 ,可能是由Leao播散性抑制引起 ,并与细胞内外信号转导和细胞凋亡有关。     

大鼠弥漫性脑损伤后脑组织中MMP-9的表达与神经元损伤

目的探讨大鼠弥漫性脑损伤后基质金属蛋白酶-9（MMP-9）在脑组织中的表达与神经元损伤的关系。方法荧光实时定量RT—PCR和免疫组化分别测定大鼠弥漫性脑损伤后MMP-9 mRN和MMP-9在脑组织中的表达。光镜和电镜观察神经元的变化。结果外伤后3h大鼠脑组织中MMP-9的阳性表达率开始升高，72h达高峰（50．35％±7．27％），伤后6h～7d各组均明显高于假手术对照组（P〈0．05）；随后开始下降，14d降至对照组水平。MMP-9 mRNA于伤后1h表达水平开始升高，72h达最高（20．56±1．29），伤后12h～7d时明显高于假手术对照组（P〈0．01）；随后开始下降，14d降至对照组水平。光镜及电镜下均可见弥漫性神经元变性和坏死，伤后72h损伤表现最为严重。结论本研究提示大鼠弥漫性脑损伤后MMP-9表达升高并可能参与了外伤后炎症反应和神经元损伤的病理过程。     

bFGF、VEGF在大鼠创伤海马脑组织中的表达

目的 研究碱性成纤维细胞生长因子(bFGF)和血管内皮细胞生长因子(VEGF)在大鼠创伤海马组织中不同时间的表达及其它们之间的关系，从分子水平探讨颅脑损伤后的病理机制，为临床治疗脑损伤的新途径提供实验基础。方法 改进Marmarou大鼠加速弥漫性脑损伤模型，取海马区创伤脑组织免疫组化染色观察bFGF、VEGF基因表达情况。结果 海马CAl区伤后6h，bFGF基因表达增加，12h达高峰；VEGF基因表达伤后24h达高峰，72h回复到对照水平。结论 bFGF、VEGF基因表达与脑损伤密切相关，作为生长因子，bFGF、VEGF可能参与颅脑损伤后神经元保护及损伤后修复过程。     

大鼠颅脑创伤后肝细胞生长因子表达变化的实验研究

目的 探讨肝细胞生长因子(HGF)在颅脑创伤后的表达趋势,为颅脑创伤治疗中的HGF干预策略提供前期研究基础. 方法 96只wistar大鼠按随机数字表法分为实验组和假手术组,实验组为液压冲击中度颅脑创伤大鼠,并分为伤后2h、6h、12h、24 h、72h、168 h、336 h组,假手术组不致伤,每组再分为两个亚组.每亚组6只,一组行HE及免疫组化染色,观察伤后病理变化及HGF的表达部位和表达量,另外一组用RT-PCR的方法 观察创伤后HGF mRNA表达情况.结果 在创伤后的大鼠大脑皮层组织中,HGF在蛋白水平以及基因水平都出现表达增高的情况.创伤边缘区HGF阳性细胞数从伤后24 h开始增多,168 h达高峰,336 h有所下降,但仍高于伤前水平,差异有统计学意义(P<0.05).HGF mRNA表达量从创伤后72 h开始增加,168 h达高峰,与假手术组比较差异有统计学意义(P<0.05). 结论 HGF作为神经营养因子和血管生长因子,可能参与了颅脑创伤后神经元的保护和组织的修复、再生.     

颅脑损伤患者血清中脑红蛋白表达变化的研究

目的观察脑红蛋白在颅脑损伤患者血清中的表达变化,探讨脑红蛋白在颅脑损伤中的作用。方法酶联免疫吸附实验(ELISA)法检测5例正常体检患者,18例颅脑损伤(据GCS评分分为3组:轻型组(GCS 13~15分),中型组(GCS 9~12分)重型组(GCS 3~8分)患者损伤后3 h、6 h、12 h、24 h、48 h、72 h患者血清中脑红蛋白(NGB)浓度。结果 1中、重度颅脑损伤组血清NGB表达明显高于对照组,轻度颅脑损伤与对照组之间无统计学差异。2血清中NGB含量在损伤后3 h处于高水平,其后逐渐下降,12 h后逐渐增高,至24 h略有升高,而后逐渐下降。颅脑损伤轻、中、重度组间均存在统计学差异(P<0.05)。结论颅脑损伤诱导脑红蛋白的表达,发挥其脑保护作用,NGB血清学水平与神经细胞受损的程度相关,受损情况越重,脑红蛋白在血清中的含量越高。     

Neuroglobin expression in rats after traumatic brain injury

In this study,we used a rat model of severe closed traumatic brain injury to explore the relationship between neuroglobin,brain injury and neuronal apoptosis.Real-time PCR showed that neuroglobin mRNA expression rapidly increased in the rat cerebral cortex,and peaked at 30 minutes and 48 hours following traumatic brain injury.Immunohistochemical staining demonstrated that neu-roglobin expression increased and remained high 2 hours to 5 days following injury.The rate of in-crease in the apoptosis-related Bax/Bcl-2 ratio greatly decreased between 30 minutes and 1 hour as well as between 48 and 72 hours post injury.Expression of neuroglobin and the anti-apoptotic factor Bcl-2 greatly increased,while that of the proapoptotic factor decreased,in the cerebral cortex post severe closed traumatic brain injury.It suggests that neuroglobin might protect neurons from apoptosis after traumatic injury by regulating Bax/Bcl-2 pathway.     

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.     

体外培养原代神经元损伤模型的建立及损伤后脑红蛋白的表达

目的建立原代神经元机械性损伤的体外细胞模型并探讨神经元损伤后脑红蛋白（NGB）的表达变化。方法取出生12h内Wistar鼠的大脑皮质，培养10-12d后，采用所设计的标准模板，直视下进行损伤，采用免疫组化方法和图像分析技术观测损伤前、后不同时间点神经元的NGB免疫组化反应及其阳性信号的灰度值。结果皮质神经元损伤2h后，NGB阳性信号强度即开始增加，16h后达高峰，随后逐渐下降，128h后恢复至损伤前水平。结论皮质神经元体外损伤模型可用于在体外观察神经元受到机械性损伤后细胞内蛋白表达变化的一些分子事件，简单实用。机械性损伤可以诱导神经元NGB在损伤后一定时间段内表达上调。     

Occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema

The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney＇s free falling method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve cell edema, degenera- tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradually de- creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradually increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.     

Expression of Nogo-A mRNA after injury of the rat central nervous system

BACKGROUND： Nogo protein has been identified as an inhibitor of axonal growth, which was highly expressed in central nervous system; however, there are only a few studies on changes of Nogo-A expression following central nervous system injury. OBJECTIVE： To investigate the dynamic expression of Nogo-A mRNA after rat central nervous system injury. DESIGN： Randomized controlled animal study. MATERIALS： Thirty-five rats were randomly divided into two groups, normal animal group （n = 5） and model group （n = 30）. The model group was then divided into six subgroups at six time points： 12, 24 hours and 3, 9, 15, and 21 days post-injury, with five rats in each subgroup. METHODS： The left parietal lobe of rats was contused by free-fall strike, and total RNA was extracted from the entire brain tissue. Semi-quantitative RT-PCR was used to detect Nogo-A mRNA expression, and the ratio between expression of the target gene and glyceraldehyde phosphate dehydrogenase was used to determine the relative expression level. MAIN OUTCOME MEASURES： To determine whether Nogo-A mRNA expression was higher than usual following brain injury. RESULTS： The level of Nogo-A mRNA started to increase 12 hours after injury （P 〈 0.05） and decreased slightly by 24 hours post-injury. Expression increased again on day 3 and reached a peak on day 9. Nogo-A mRNA expression started to decrease on day 15, and then decreased to normal levels at days 21 （P 〉 0.05）. CONCLUSION： After injury of the central nervous system, Nogo-A may play a pivotal role in obstructing regeneration of the nerve.     

Calcium overloading in traumatic axonal injury by lateral head rotation: a morphological evidence in rat model.

The study investigated morphologically axonal calcium overloading and its relationship with axonal structural changes. Twelve SD rats were divided into an injury and a sham group. The rat model of traumatic axonal injury (TAI) by lateral head rotation was produced. The oxalate-pyroantimonate technique for calcium localization was used to process the rat's medulla oblongata tissues with thin sections observed electron-microscopically for axonal structure and calcium precipitates on it. The axonal damage in medulla oblongata appeared at 2 h post-injury, gradually became diffuse and severe, and continued to exist at 24 hours. At 2 hours, calcium precipitates were deposited on separated lamellae and axolemma, but were rarely distributed in the axoplasm. At 6 hours, calcium precipitates occurred on separated lamellae and axolemma in much higher density, but on axoplasm in extremely small amounts. Some axons, though lacking structural changes of the myelin sheath, sequestered plenty of calcium deposits on their swollen mitochondria. At 24 hours, damaged axons presented with much more severe lamellae separation and calcium deposits. Axonal calcium overloading developed in rat TAI model using lateral head rotation. This was significantly related to structural damage in the axons. These findings suggest the feasibility of using calcium antagonists in cope the management of human DAI in its very early stage.     

Resuscitation therapy for traumatic brain injuryinduced coma in rats:mechanisms of median nerve electrical stimulation

In this study, rats were put into traumatic brain injury-induced coma and treated with median nerve electrical stimulation. We explored the wake-promoting effect, and possible mechanisms, of median nerve electrical stimulation. Electrical stimulation upregulated the expression levels of orexin-A and its receptor OX1 R in the rat prefrontal cortex. Orexin-A expression gradually increased with increasing stimulation, while OX1 R expression reached a peak at 12 hours and then decreased. In addition, after the OX1 R antagonist, SB334867, was injected into the brain of rats after traumatic brain injury, fewer rats were restored to consciousness, and orexin-A and OXIR expression in the prefrontal cortex was downregulated. Our findings indicate that median nerve electrical stimulation induced an up-regulation of orexin-A and OX1 R expression in the prefrontal cortex of traumatic brain injury-induced coma rats, which may be a potential mechanism involved in the wake-promoting effects of median nerve electrical stimulation.     

Wake-promoting effects of vagus nerve stimulation after traumatic brain injury: upregulation of orexin-A and orexin receptor type 1 expression in the prefrontal cortex

Orexins, produced in the lateral hypothalamus, are important neuropeptides that participate in the sleep/wake cycle, and their expression coincides with the projection area of the vagus nerve in the brain. Vagus nerve stimulation has been shown to decrease the amounts of daytime sleep and rapid eye movement in epilepsy patients with traumatic brain injury. In the present study, we investigated whether vagus nerve stimulation promotes wakefulness and affects orexin expression. A rat model of traumatic brain injury was established using the free fall drop method. In the stimulated group, rats with traumatic brain injury received vagus nerve stimulation(frequency, 30 Hz; current, 1.0 mA; pulse width, 0.5 ms; total stimulation time, 15 minutes). In the antagonist group, rats with traumatic brain injury were intracerebroventricularly injected with the orexin receptor type 1(OX1R) antagonist SB334867 and received vagus nerve stimulation. Changes in consciousness were observed after stimulation in each group. Enzyme-linked immunosorbent assay, western blot assay and immunohistochemistry were used to assess the levels of orexin-A and OX1R expression in the prefrontal cortex. In the stimulated group, consciousness was substantially improved, orexin-A protein expression gradually increased within 24 hours after injury and OX1R expression reached a peak at 12 hours, compared with rats subjected to traumatic brain injury only. In the antagonist group, the wake-promoting effect of vagus nerve stimulation was diminished, and orexin-A and OX1 R expression were decreased, compared with that of the stimulated group. Taken together, our findings suggest that vagus nerve stimulation promotes the recovery of consciousness in comatose rats after traumatic brain injury. The upregulation of orexin-A and OX1R expression in the prefrontal cortex might be involved in the wake-promoting effects of vagus nerve stimulation.     

Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND: Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells.OBJECTIVE: To investigate the effects of valproic acid on proliferation of endogenous neural sterm cells in a rat model of spinal cord injury.DESIGN, TIME AND SETTING: A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007.MATERIALS: A total of 45 adult, Wistar rats were randomly divided into sham surgery (n=5), injury(n=20), and valproic acid (n=20) groups. Valproic acid was provided by Sigma, USA.METHODS: Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours (twice in total).MAIN OUTCOME MEASURES: Nestin expression (5 mm from injured center) was detected using immunohistochemistry at 1, 3 days, 1, 4, and 8 weeks post-injury.RESULTS: Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week (P<0.05).Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was nosignificant difference in nestin expression between the valproic acid and injury groups. At 1 week,there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group (P<0.05). Expression reached a peak by 4 weeks, and it was still present at 8 weeks.CONCLUSION: Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.