深低温停循环中脑红蛋白表达及其脑损伤防护意义实验研究
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摘要
深低温停循环(Deep hypothermic circulatory arrest，DHCA)技术可以提供一个无血而宁静的手术环境，是部分婴幼儿先天性心脏病及成人复杂大血管疾病外科治疗中的重要手段。近年来，随着心、脑、肺、肝、肾等保护技术的改进、多学科合作增加，DHCA的应用日益广泛。脑对缺血缺氧最敏感，同时它对缺血缺氧的耐受性也最差。深低温下脑代谢依然存在，长时间DHCA常导致不同程度的神经系统损害。DHCA脑损害的机制复杂，目前停循环后脑保护研究国内外均主要集中在如何降低脑代谢上，而从脑氧代谢的机制出发，研究增加氧贮备、转移和利用才是解决问题的根本办法。近年脑氧代谢基础研究取得重大发展。
2000年德国Burmester等发现人和小鼠脑内特异的携氧球蛋白—脑红蛋白(neuroglobin，Ngb)，为脑缺氧的研究提供了全新思路。人Ngb基因为保守性较高的单拷贝基因，定位于染色体14q24，全长cDNA序列共1909bp，编码151个氨基酸。Ngb主要表达于脊椎动物神经元的细胞质，与氧有很高的亲和力，功能上类似于肌红蛋白，可特异性地为脑供氧，在分子水平调节脑组织氧供应状态。作为一个重要的内源性神经保护因子，Ngb在组织中表达水平的高低与组织的缺氧耐受性呈正相关。脊椎动物脑红蛋白可能的功能有：1贮存氧，生理情况下与氧充分氧合达饱和状态，缺氧时释放氧。2氧的传递转运氧通过血脑屏障，加速氧向神经元中的线粒体扩散。3作为一种缺氧感受器，保护下游缺氧损害。4清除毒性物质如NO。氯化高铁血红蛋白(Hemin)，又名正铁血红素、血晶素，长久以来，它是一个被用于贫血治疗的药物，早在1983年美国FDA就批准其用于临床治疗。近年来，Hemin在其他各系统中的保护作用逐渐得到重视。实验证实，Hemin对离体Ngb表达呈显著增强作用，且呈浓度和时间依赖性。Hemin作为血红素氧合酶(HO)的底物和诱导剂，可诱导脑红蛋白表达，抵抗自由基损伤，减少兴奋性氨基酸毒性，在心、脑、肠等器官的缺血缺氧损伤中具有重要保护作用。
随着分子生物学等技术在医学中的广泛应用，人们对脑损伤的病理机制认识不断加深，自由基、一氧化氮、细胞因子及粘附分子、蛋白类生化标志物等在体外循环及DHCA脑损伤中的作用越来越受到人们的关注。1)S—100β蛋白由Moore于1965年在牛脑中发现，是一种相对分子质量为21×10~3的酸性钙结合蛋白，浓度特异性地存在于中枢神经系统。脑损伤后脑S—100β蛋白过度表达和释放，并迅速释放入血液。由于它的半衰期短，血S—100β蛋白水平在伤后短时间内迅速下降。当脑损伤后胶质细胞迟发性功能障碍或持续死亡时可引起S—100β蛋白外溢，继发性脑损害使血脑屏障进一步破坏，随着病情的发展，可能出现继发性升高或持续高值，表明进行性继发性脑损害。2)髓鞘碱性蛋白(myelin base protein，MBP)：神经组织髓鞘碱性蛋白主要位于髓磷脂浆膜面，与髓鞘脂质紧密结合，维持中枢神经系统髓鞘结构和功能的稳定，在神经纤维中起着绝缘和快速传导的作用。当中枢神经系统髓鞘破坏时，髓鞘碱性蛋白可释放入中枢神经系统和血中，最后降解经尿排出。研究资料表明，髓鞘碱性蛋白与急慢性脑血管病、实验性变态反应性脑脊髓炎、多发性硬化及其他许多神经疾病有关，是反映中枢神经系统有无实质性损害，特别是无髓鞘脱失的一个较特异的生化指标，还可作为评价治疗及预后的指标之一。3)肿瘤坏死因子α(TNFα)：TNFα是近年来研究较深入的由多种细胞产生和分泌的一种细胞因子，研究提示TNFα在缺血性脑损伤病理机制方面起着双重效应。有害效应是指TNFα可加重脑缺血后神经元的损伤。相反，有人认为TNFα可能具有神经元保护机能。由于缺乏TNFα在脑缺血损伤过程中产生有利效应的大量系统研究，多数学者认为它主要作为炎性介质参与损伤反应。这为脑缺血的治疗学研究指明了新的方向。4)血管细胞间黏附分子—1(VCAM-1)：属于黏附分子的免疫球蛋白超家族存在于血管内皮细胞膜上，受致炎因子刺激时表达增加，参与淋巴细胞的活化、迁移和造血细胞的生长发育，并在炎症、肿瘤转移等病理过程中起重要作用。相关研究表明VCAM—1在脑缺血炎症发生、发展过程中起着关键作用，调控脑缺血时VCAM—1的表达及作用可减轻脑缺血症状，保护脑缺血损伤。
本实验利用实时荧光PCR定量分析脑红蛋白mRNA在体外循环及深低温停循环(DHCA)情况下的表达，并通过检测血浆脑损伤相关指标S-100β、MBP、TNFα和VCAM-1等的变化，探讨脑红蛋白及相关脑损伤指标在DHCA中脑损伤诊断、治疗及预后的意义及机制。
目的：
检测空白对照组与Hemin处理组对犬DHCA不同时期Ngb mRNA表达以及血浆S—100β蛋白、MBP、TNFα、VCAM-1及脑皮质超微结构的改变推断脑红蛋白及相关指标在DHCA早期脑保护中的作用及机制，以探索预防和治疗DHCA脑损害的方法。
方法：
1、采用成年犬DHCA模型，选择健康成年杂种犬10只，随机分为2组。对照组(Ⅰ组)；Hemin处理组(Ⅰ组)：实验前24小时给予Hemin 50mg／kg静脉注射预处理。实验犬以3％戊巴比妥钠诱导麻醉，气管插管，呼吸机辅助呼吸。左颈静脉逆行插管取脑静脉血，左颈动脉插管测平均动脉压(MBP)。右侧开颅取脑皮质。主动脉插管及上、下腔插管建立体外循环转流降温至鼻咽部温18℃停循环。90min后恢复循环、复温再灌注至鼻咽部温37℃后停机，停机60min后放血处死实验动物。
2、各组分别于体外循环0min、停循环时(0min)、停循环60min、复温再灌注45min抽取颈静脉测S—100β蛋白、MBP、TNFα、VCAM-1。分别于上述各时间点取脑皮质迅速冻存，待全部动物实验结束后统一行实时荧光PCR定量检测Ngb mRNA的表达情况。复温再灌注60min在颈动脉灌注4％多聚甲醛固定，取脑皮质行透射电镜观察脑组织超微结构。
3、实验数据以SPSS11.5统计软件处理，重复测量的方差分析及单因素方差分析数据对比，Bonfferoni法两两比较。两组间各点比较用两样本t检验，以P≤0.05为具有统计学意义。
结果：
1、体外循环后特别是降温后脑红蛋白Ngb mRNA的拷贝均升高，至DHCA 60min达最高值((?)±S=565680681.56±223521679.77 Copy Number／ug)，复温再灌注后轻微下降(图3-1)。不同时间点脑红蛋白Ngb mRNA的拷贝浓度间差异有显著性意义(F=16.745，P＜0.001)。实验组和对照组对脑红蛋白Ngb mRNA的拷贝浓度影响差异无显著性意义(F=5.153，P=0.392)。分组与各时间点脑红蛋白Ngb mRNA的拷贝浓度无交互效应(F=0.249，P=0.862)。各时间点脑红蛋白Ngb mRNA的拷贝浓度随时间变化趋势不同，降温至停循环期间脑红蛋白Ngb mRNA的拷贝数开始升高，至DHCA60min时拷贝数达到最高，复温再灌注45min后脑红蛋白Ngb mRNA的拷贝数较DHCA60min时略有下降，但仍高于DHCA开始时。DHCA60min时以及复温再灌注45min后脑红蛋白Ngb mRNA的拷贝数均显著高于体外循环开始前及DHCA开始时(P＜0.048)。
2、体外循环后犬血浆S-100β蛋白浓度明显升高，从循环开始至DHCA时增长幅度较高，随着DHCA的时间延长以及复温再灌注后血浆S-100β蛋白浓度仍继续升高，但增长幅度变缓。不同时间点血浆S-100β蛋白浓度间差异有显著性意义(F=1114.099，P＜0.001)。实验组和对照组对血浆S-100β蛋白浓度影响差异无显著性意义(F=0.599，P=0.461)。分组与各时间点血浆S-100β蛋白浓度无交互效应(F=0.288，P=0.678)。各时间点血浆S-100β蛋白浓度随时间变化趋势不同，从循环开始至DHCA时血浆S-100β蛋白浓度影响最大(P＜0.001)。复温再灌注45min后血浆S-100β蛋白浓度最高((?)±S=17.59±0.35 ng／ml)，但与DHCA60min时差异无显著性意义(P=0.289)。
3、体外循环后犬血浆MBP浓度缓慢升高，至DHCA60分钟，犬循环恢复开始重新灌注时，血浆内MBP浓度明显升高达最高值((?)±S=4.17±0.88 ng／ml)，在复温再灌注45分钟时血浆MBP浓度又明显下降甚至低于体外循环开始时。实验组和空白组不同时间点血浆MBP浓度有统计学差异(F=44.999，P＜0.001)，实验组与空白组间无统计学差异(F=1.988，P=0.196)，不同时间点血浆MBP浓度与分组之间无交互效应(F=1.081，P=0.376)。前二个时间点，犬血浆MBP浓度变化不大并没有明显的差异，但DHCA结束恢复脑灌注时犬血浆MBP浓度与前二个时间点有统计学差异(P＜0.001)，当脑复温灌注一段时间后，至复温再灌注45分钟时血浆MBP浓度又恢复甚至略低于体外循环开始时水平，与DHCA60分钟，犬脑刚恢复灌注时有统计学差异(P=0.001)。
4、体外循环后犬血浆TNFα浓度缓慢下降，至DHCA 60分钟，犬循环恢复开始重新灌注时，血浆内犬TNFα浓度达到实验最低值((?)±S=316.17±3.07ng／ml)，在复温再灌注45分钟时血浆TNFα浓度又缓慢上升，但没有恢复到体外循环前的水平，不同时间点间差异虽有显著性差异(F=4.258，P=0.015)，但进行不同时间点的多重比较(即两两时间比较)后发现各时间的TNFα浓度变化没有统计学差异(P＞0.152)。并且空白对照组与实验组无显著性意义(F=1.108，P=0.323)。不同时间点血浆TNF-α浓度与分组无交互效应(F=0.614，P=0.613)。
5、体外循环后犬血浆VCAM-1浓度逐渐升高，至复温再灌注45min达最大值((?)±S=35.14±3.26ng／ml)。两组间不同时间点血浆VCAM-1浓度有显著性差异(F=15.838，P=0.001)，两组间血浆VCAM-1浓度无显著性差异(F=0.525，P=0.489)。不同时间点血浆VCAM-1浓度与分组无交互效应(F=0.077，P=0.972)。在实验的前三个时间段，犬血浆VCAM-1浓度变化不大并没有明显的差异，但从DHCA60分钟至复温再灌注45分钟时段中增长幅度较高，并产生统计学差异(P=0.013)。即在犬脑部温度逐渐恢复后，外周血重新灌注后一段时间血浆中的VCAM-1浓度开始明显增高。
6、从Ngb mRNA表达与相关脑损伤指标的相关分析可得出：Ngb mRNA的表达变化与血浆S-100β蛋白在DHCA过程中存在正相关关系(偏相关系数为：0.4603，P=0.004)，相关较密切。
7、脑组织超微结构变化：
DHCA及复温再灌注后脑皮质神经元细胞超微结构改变比较明显，主要表现为线粒体肿胀明显，部分呈气球样变，线粒体嵴减少、脱落；细胞膜连续性完整，双层结构显示不清；溶酶体增生明显，并可见吞噬的线粒体。对照组与实验组脑超微结构无明显差异。
结论：
1、Hemin预处理可提高DHCA不同时期Ngb mRNA的表达，但由于拷贝数量级较大，统计学差异并不显著(F=5.153，P=0.053)。实验组与对照组相比，对改善DHCA期脑损伤的作用并不明显。氯化高铁血红素和缺氧所诱导的NGB表达存在不同机制，并且Hemin诱导的脑红蛋白mRNA表达明显慢于缺氧诱导的表达。
2、由于目前尚不能显著刺激Ngb mRNA表达，故脑红蛋白在DHCA条件下的脑保护作用并不明显，其机理及作用途径仍需进一步研究。
3、血浆S-100β蛋白、MBP、VCAM-1可作为DHCA条件下脑损伤早期的敏感指标，其中以S-100β蛋白、VCAM-1为佳，MBP次之。TNFα并不适宜作为DHCA条件下脑损伤早期的敏感指标，其在DHCA条件下脑损伤的双重作用仍需进一步探讨。
4、从Ngb mRNA表达与相关脑损伤指标的相关分析可得出，脑红蛋白有望成为DHCA条件下脑损伤早期一种新的敏感指标。其在脑损伤的诊断、预后和保护作用有待于更多的实验和临床研究。
5、脑损伤后血液中神经系统相关蛋白质时间—浓度曲线关系，综合分析多种标志物动态变化比检测单一神经系统相关蛋白质的含量变化对判断脑损伤程度、评估预后、调整治疗方案等更有临床意义。
Various techniques for the heart, brain, lung, liver, and kidney protection have been developed and with increasing cooperation between researchers from different fields, deep hypothermic circulatory arrest (DHCA) has gained more extensive clinical application. Currently, DHCA has become a very important means for surgical management of some congenital cardiac defects in infants as well as of complicated major blood vessel abnormalities in adults. Among the vital organs of the body, the brain is especially vulnerable to anoxia and ischemia. Hypothermia allows chances for the organs to evade from disastrous damages in the context of circulatory arrest by significantly lower the metabolic rate, but even so, prolonged DHCA still results in nerve system impairment of various degrees. The mechanism of DHCA-induced brain injury is complex, and most researches on brain protection following circulatory arrest give their primary attention to the means of lowering brain metabolism, which, however, does not seem to provide a solution for all the problems. A typical solution for brain protection may lie in the exploit of the mechanism of brain oxygen metabolism, so as to develop methods to promote brain oxygen preservation, transport and economic consumption. Basic studies of brain oxygen metabolismhave made great progress in recent years.
In 2000, Burmester et al discovered an oxygen-carrying globulin specific to the brain of human and mice, and gave it the name neuroglobin (Ngb), which shed light on a new scope of cerebral anoxia research. As a highly conservative single-copy gene in human, Ngb gene is located on chromosome 14q24 with a full cDNA length of 1909 bp that encodes 151 amino acids. Ngb is mainly expressed in the neuronal cytoplasm of vertebrates with high affinity to oxygen and functionally resembles hemoglobin in that it provides oxygen supply specifically to the brain and modulates the oxygen supply of the brain at the molecular level. As an important endogenous neuroprotective factor, the expression level of Ngb is positively associated with the tolerance of anoxia of the brain tissue. Ngb probably performs the functions of (1) oxygen preservation, in that it binds to oxygen in physiological conditions and release oxygen in anoxia, (2) oxygen transport, it carries oxygen through the blood-brain barrier and accelerates the diffusion of the oxygen into the mitochondria in the neurons, (3) anoxia sensors, which protects from downstream anoxic damages, and (4) clearing toxic substances such as NO.
For a long time Hemin has been used for treatment of anemia but recently, its protective role in other systems is given increasing attention. Experimental evidences have proved that Hemin can significantly enhance Ngb expression in vitro in a dose- and time-dependent manner. As a substrate and inducer of heme oxygenase (HO), Hemin can induce the expression of Ngb, resist free radical injury and reduce the toxicity of excitatory amino acids, thus provides important protection of the heart, brain, and guts against ischemic and anoxic injuries.
As techniques of molecular biology are widely applied in medical science research, increasing understanding of the pathological basis of brain injury has been obtained, and the roles of free radicals, NO, cytokines and adhesion molecules, and protein biochemical markers in extracorporeal circulation and DHCA have attracted increasing attention.
S-100β, a protein found in bovine brain by Moore in 1965 with relative molecular mass of 21×103, exists concentration-dependently in the central nervous system as an acidic calcium-binding protein. S-100βis overexpressed in the brain tissue and rapidly released into the circulation following brain injury, but due to its very short half-life, the level of this protein in the blood soon declines. Delayed functional impairment or progressive death of the glial cells following brain injury leads to S-100βleakage, and secondary brain injury causes further damage of the blood-brain barrier. As the condition worsens, secondary elevation of S-100βor persistent presence of high-level S-100βmay occur, which suggests progressive secondary brain injury.
Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons and binds tightly with myelin to maintain the integrity and functional stability of the neural sheath. In the event of neural sheath destruction, MBP can be released in the nervous system and blood circulation, and finally degraded and cleared through urination. Current data suggest that MBP is associated with acute or chronic cerebrovascular diseases, experimental allergic encephalomyelitis, multiple sclerosis and many other neurological diseases, and considered a specific biomedical indicator of organic damage of the nervous system, particularly demyelination. MBP also serves the purpose of therapeutic effect evaluation and prognostic assessment.
Tumor necrosis factorα(TNFα) is a extensively studied cytokine secreted by various types of cells. Studies suggest that this cytokine plays a double-faced role in ischemic brain injury in that TNFαworsens neuronal damage following ischemic brain injury but may also execute neuroprotective functions. But due to the insufficiency of systemic research data demonstrating the beneficial effect of TNFαin ischemic brain injury, most researchers inclined to believe that TNFαparticipates in the pathological process of brain injury as an inflammatory agent. This seems to provide new insight into therapy of ischemic brain injury.
Vascular cell adhesion molecule-1 (VCAM-1), a member of the adhesion molecule immunoglobulin superfamily, is located on the vascular endothelial cell membrane. Stimulation with inflammatory agents results in increased expression of VCAM-1 which participates in lymphocyte activation and migration and the growth and development of hemopoietic cells and is also involved in such pathological processes as inflammation and tumor metastasis. Researches have identified important roles of VCAM-1 in the inflammatory response in relation to cerebral ischemia and its progression. VCAM-1 expression can alleviate the symptoms of brain ischemia and protect against ischemic brain injury.
In this study, real-time fluorescence quantitative PCR was employed to analyze Ngb mRNA expression during extracorporeal circulation and DHCA in relation to the expressions of S-100β, MBP, TNFαand VCAM-1. The value of Ngb and other indices related to brain injury was explored in the diagnosis, therapy and prognostic assessment of brain injury due to DHCA.
Objective:
Ngb expression and plasma levels of S-100β, MBP, TNFαand VCAM-1 were detected at different time points during DHCA in dogs with or without Hemin treatment. Ultrastructural changes of the cerebral cortex was also observed to reveal the role and mechanism of Ngb and the other factors in protection against early-stage brain injury due to DHCA, thereby finding effective means for prevention and therapy of brain injury due to DHCA.
Methods:
Ten healthy hybrid dogs were randomized into control group and Hemin treatment group, and in the latter, the dogs were given 50 mg/kg Hemin 24 hours prior to the experiment. The dogs were anesthetized with 3% sodium pentobarbital followed by tracheal intubation with artificial ventilation. Cannulation of the left jugular vein was performed to collect cerebral venous blood sample, and the left carotid artery was also cannulated to measure the mean arterial blood pressure (MBP). Craniotomy was performed on the right side of the skull to obtain cerebral cortex sample. Cannulation of the aorta and the inferior and superior vena cava was performed to establish extracorporeal circulation, which was terminated till the nasopharyngeal temperature dropped to 18℃. Ninety minutes later, the circulation was restarted and terminated till a nasopharyngeal temperature of 37℃. One hour after extracorporeal circulation termination, the dogs were killed by blood depletion.
Venous blood samples were obtained from the left jugular vein immediately after extracorporeal circulation establishment, 0 and 60 minutes after extracorporeal circulation termination, and 45 minutes of reperfusion, respectively, for measurement of S-100β, MBP, TNFα, and VCAM-1 levels. Cortical samples were also obtained at these time points and frozen for real-time fluorescence quantitative PCR for Ngb mRNA measurement. At 60 minutes of reperfusion, 4% paraformaldehyde was perfused through the carotid artery and cerebral cortex samples were harvested for transmission electron microscopic observation of the ultrastructures.
The data of the measurement were statistically analyzed with SPSS11. 5 software by repeated measure analysis of variance and one-way ANOVA. Bonfferoni test was performed for paired comparison. A P value no greater than 0.05 was considered to indicate significant difference.
RESULTS
1、The establishment of extracorporeal circulation resulted in increased Ngb mRNA expression, which was especially obvious after body temperature reduction, and at 60 min after DHCA its expression reached the peak level((?)±S=565680681.56±223521679.77Copy Number/ug), followed by slight decrease after body temperature recovery. The concentration of Ngb mRNA showed significant differences between different time points (F=16.745, P＜0.001), but were comparable between the Hemin and control groups (F=5.153, P=0.392). There is no crossover effect of the concentration of Ngb mRNA between groups and different time points (F=0.249, P=0.862). At each time points of measurement, Ngb mRNA concentration exhibited different patterns of alteration, specifically, it increased after body temperature reduction till circulation arrest, reaching the peak level at 60 minutes of DHCA and a slight reduction was noted at 45 minutes of reperfusion when Ngb mRNA level was still higher than that measured at the initiation of DHCA. Ngb mRNA level at 60 minutes of DHCA and at 45 minutes of reperfusion was significantly higher than that measured before extracorporeal circulation establishment and at the start of DHCA (P＜0.048).
2、Following extracorporeal circulation establishment plasma S-100βprotein level showed significant increment, which was most obvious within the period between extracorporeal circulation initiation and DHCA. As DHCA was prolonged, S-100βprotein level kept increasing even after reperfusion, but the increment showed a gradually lowered magnitude. S-100βprotein level showed significant differences between the time points of measurement (F=1114.099, P＜0.001). But between Hemin and control groups, S-100βprotein levels showed no significant difference (F=0.599, P=0.461). There is no crossover effect of S-100βprotein level between groups and different time points (F=0.249, P=0.862). Most obvious changes of S-100βprotein level was noted in the period between extracorporeal circulation initiation and DHCA (P＜0.001). The highest S-100βprotein level occurred at 45 minutes of reperfusion((?)±S=17.59±0.35 ng/ml), but this high level showed no significant difference from that measured at 60 minutes of DHCA (P=0.289).
3、Plasma MBP level increased gradually following extracorporeal circulation initiation till 60 minutes of DHCA, and at reperfusion, plasma BMA reached its peak level ((?)±S=4.17±0.88 ng/ml), but at 45 minutes of reperfusion, MBP level underwent a significant reduction even to a level below that before extracorporeal circulation initiation. MBP level showed significant differences between the time points of measurement (F=44.999, P＜0.001). But between Hemin and control groups, MBP level showed no significant difference (F=1.988, P=0.196). There is no crossover effect of MBP level between groups and different time points (F=1.081, P=0.376). Between extracorporeal circulation initiation and 60 minutes of DHCA, plasma DHCA level did not undergo significant changes until the termination of DHCA and initiation of reperfusion (P＜0.001), but at 45 minutes of reperfusion, BMP level recovered and became even lower than the level before extracorporeal circulation, showing significant difference from the level at 60 minutes of DHCA (P=0.001).
4、Plasma TNFαlevel was slowly reduced after extracorporeal circulation initiation till 60 minutes of DHCA, and at the time of reperfusion, TNFαlevel reached its lowest ((?)±S=316.17±3.07 ng/ml), followed by slow increase at 45 minutes of reperfusion but failed to recover its former level. Its level showed significant differences between the time points (F=4.258, P=0.015), but these differences were not supported statistically after multiple comparison (P＞0.152). TNFαlevel was not significantly different between the control and Hemin groups (F=1.108, P=0.323). There is no crossover effect of TNFαlevel between groups and different time points (F=0.614, P=0.613).
5、Plasma VCAM-1 level increased gradually following extracorporeal circulation initiation and at 45 minutes of reperfusion, plasma VCAM-1 level reached its peak level ((?)±S=35.14±3.26ng/ml). Plasma VCAM-1 level showed significant differences between the time points of measurement (F=15.838, P=0.001). But between Hemin and control groups, Plasma VCAM-1 level showed no significant difference (F=0.525, P=0.489). There is no crossover effect of Plasma VCAM-1 level between groups and different time points (F=0.077, P=0.972). In the former 3 stages of the experiment, VCAM-1 level did not undergo significant alterations, but from 60 minutes of DHCA to 45 minutes of reperfusion, VCAM-1 level showed obvious increase (P=0.013). In other words, VCAM-1 level began to increase obviously following reperfusion after brain temperature recovery.
6、Correlation analyses between Ngb mRNA expression and the brain injury indices described above revealed that Ngb mRNA expression was in close positive correlation with plasma S-100βlevel during DHCA (with coefficient of partial correlation of 0.4603, P=0.004).
7、After DHCA and reperfusion, obvious changes were observed in the ultrastructure of the cortical neurons, including swelling of the mitochondria and reduction of the mitochondrial crest. The cell membrane remained continuous and intact with clear bilayer structure. Lysosome proliferation was obvious, and phagocytosed mitochondria could be seen. These changes were not significantly different between the control and Hemin groups.
CONCLUSION
Hemin pretreatment may enhance Ngb mRNA expression at different phases of DHCA, but due to the large magnitude of the mRNA copies, no statistically significant differences are produced (F=5.153, P=0.053). Hemin failed to show obvious effect in improving brain injury during DHCA in comparison with the control group. Hemin and anoxia induce Ngb expression through different mechanisms, and the former seems to induce Ngb expression at a much lower pace than the latter.
As currently no effective means are available to sufficiently stimulate Ngb expression, the protective effect of Ngb against brain injury is not obvious during DHCA, and the mechanism needs further exploration.
Plasma S-100βprotein, MBP, and VCAM-1 can be sensitive indicators of early brain injury during DHCA, but S-100βprotein and VCAM-1 serve this purpose better than MBP. TNFαdoes not seem to suit for this purpose, and its double effects in brain injury in relation to DHCA needs further investigation.
Ngb has the potential as a novel sensitive indicator for brain injury during DHCA, but its value in the diagnosis, prognostic evaluation and protective effect in brain injury awaits more extensive laboratory and clinical research.
Analysis of the time-concentration curve of the nervous system-related proteins in the blood following brain injury and comprehensive evaluation of the dynamic changes of multiple markers, other than single such proteins, can be more valuable for brain injury severity assessment, prognostic evaluation and treatment planning.
2000年德国Burmester等发现人和小鼠脑内特异的携氧球蛋白—脑红蛋白(neuroglobin，Ngb)，为脑缺氧的研究提供了全新思路。人Ngb基因为保守性较高的单拷贝基因，定位于染色体14q24，全长cDNA序列共1909bp，编码151个氨基酸。Ngb主要表达于脊椎动物神经元的细胞质，与氧有很高的亲和力，功能上类似于肌红蛋白，可特异性地为脑供氧，在分子水平调节脑组织氧供应状态。作为一个重要的内源性神经保护因子，Ngb在组织中表达水平的高低与组织的缺氧耐受性呈正相关。脊椎动物脑红蛋白可能的功能有：1贮存氧，生理情况下与氧充分氧合达饱和状态，缺氧时释放氧。2氧的传递转运氧通过血脑屏障，加速氧向神经元中的线粒体扩散。3作为一种缺氧感受器，保护下游缺氧损害。4清除毒性物质如NO。氯化高铁血红蛋白(Hemin)，又名正铁血红素、血晶素，长久以来，它是一个被用于贫血治疗的药物，早在1983年美国FDA就批准其用于临床治疗。近年来，Hemin在其他各系统中的保护作用逐渐得到重视。实验证实，Hemin对离体Ngb表达呈显著增强作用，且呈浓度和时间依赖性。Hemin作为血红素氧合酶(HO)的底物和诱导剂，可诱导脑红蛋白表达，抵抗自由基损伤，减少兴奋性氨基酸毒性，在心、脑、肠等器官的缺血缺氧损伤中具有重要保护作用。
随着分子生物学等技术在医学中的广泛应用，人们对脑损伤的病理机制认识不断加深，自由基、一氧化氮、细胞因子及粘附分子、蛋白类生化标志物等在体外循环及DHCA脑损伤中的作用越来越受到人们的关注。1)S—100β蛋白由Moore于1965年在牛脑中发现，是一种相对分子质量为21×10~3的酸性钙结合蛋白，浓度特异性地存在于中枢神经系统。脑损伤后脑S—100β蛋白过度表达和释放，并迅速释放入血液。由于它的半衰期短，血S—100β蛋白水平在伤后短时间内迅速下降。当脑损伤后胶质细胞迟发性功能障碍或持续死亡时可引起S—100β蛋白外溢，继发性脑损害使血脑屏障进一步破坏，随着病情的发展，可能出现继发性升高或持续高值，表明进行性继发性脑损害。2)髓鞘碱性蛋白(myelin base protein，MBP)：神经组织髓鞘碱性蛋白主要位于髓磷脂浆膜面，与髓鞘脂质紧密结合，维持中枢神经系统髓鞘结构和功能的稳定，在神经纤维中起着绝缘和快速传导的作用。当中枢神经系统髓鞘破坏时，髓鞘碱性蛋白可释放入中枢神经系统和血中，最后降解经尿排出。研究资料表明，髓鞘碱性蛋白与急慢性脑血管病、实验性变态反应性脑脊髓炎、多发性硬化及其他许多神经疾病有关，是反映中枢神经系统有无实质性损害，特别是无髓鞘脱失的一个较特异的生化指标，还可作为评价治疗及预后的指标之一。3)肿瘤坏死因子α(TNFα)：TNFα是近年来研究较深入的由多种细胞产生和分泌的一种细胞因子，研究提示TNFα在缺血性脑损伤病理机制方面起着双重效应。有害效应是指TNFα可加重脑缺血后神经元的损伤。相反，有人认为TNFα可能具有神经元保护机能。由于缺乏TNFα在脑缺血损伤过程中产生有利效应的大量系统研究，多数学者认为它主要作为炎性介质参与损伤反应。这为脑缺血的治疗学研究指明了新的方向。4)血管细胞间黏附分子—1(VCAM-1)：属于黏附分子的免疫球蛋白超家族存在于血管内皮细胞膜上，受致炎因子刺激时表达增加，参与淋巴细胞的活化、迁移和造血细胞的生长发育，并在炎症、肿瘤转移等病理过程中起重要作用。相关研究表明VCAM—1在脑缺血炎症发生、发展过程中起着关键作用，调控脑缺血时VCAM—1的表达及作用可减轻脑缺血症状，保护脑缺血损伤。
本实验利用实时荧光PCR定量分析脑红蛋白mRNA在体外循环及深低温停循环(DHCA)情况下的表达，并通过检测血浆脑损伤相关指标S-100β、MBP、TNFα和VCAM-1等的变化，探讨脑红蛋白及相关脑损伤指标在DHCA中脑损伤诊断、治疗及预后的意义及机制。
目的：
检测空白对照组与Hemin处理组对犬DHCA不同时期Ngb mRNA表达以及血浆S—100β蛋白、MBP、TNFα、VCAM-1及脑皮质超微结构的改变推断脑红蛋白及相关指标在DHCA早期脑保护中的作用及机制，以探索预防和治疗DHCA脑损害的方法。
方法：
1、采用成年犬DHCA模型，选择健康成年杂种犬10只，随机分为2组。对照组(Ⅰ组)；Hemin处理组(Ⅰ组)：实验前24小时给予Hemin 50mg／kg静脉注射预处理。实验犬以3％戊巴比妥钠诱导麻醉，气管插管，呼吸机辅助呼吸。左颈静脉逆行插管取脑静脉血，左颈动脉插管测平均动脉压(MBP)。右侧开颅取脑皮质。主动脉插管及上、下腔插管建立体外循环转流降温至鼻咽部温18℃停循环。90min后恢复循环、复温再灌注至鼻咽部温37℃后停机，停机60min后放血处死实验动物。
2、各组分别于体外循环0min、停循环时(0min)、停循环60min、复温再灌注45min抽取颈静脉测S—100β蛋白、MBP、TNFα、VCAM-1。分别于上述各时间点取脑皮质迅速冻存，待全部动物实验结束后统一行实时荧光PCR定量检测Ngb mRNA的表达情况。复温再灌注60min在颈动脉灌注4％多聚甲醛固定，取脑皮质行透射电镜观察脑组织超微结构。
3、实验数据以SPSS11.5统计软件处理，重复测量的方差分析及单因素方差分析数据对比，Bonfferoni法两两比较。两组间各点比较用两样本t检验，以P≤0.05为具有统计学意义。
结果：
1、体外循环后特别是降温后脑红蛋白Ngb mRNA的拷贝均升高，至DHCA 60min达最高值((?)±S=565680681.56±223521679.77 Copy Number／ug)，复温再灌注后轻微下降(图3-1)。不同时间点脑红蛋白Ngb mRNA的拷贝浓度间差异有显著性意义(F=16.745，P＜0.001)。实验组和对照组对脑红蛋白Ngb mRNA的拷贝浓度影响差异无显著性意义(F=5.153，P=0.392)。分组与各时间点脑红蛋白Ngb mRNA的拷贝浓度无交互效应(F=0.249，P=0.862)。各时间点脑红蛋白Ngb mRNA的拷贝浓度随时间变化趋势不同，降温至停循环期间脑红蛋白Ngb mRNA的拷贝数开始升高，至DHCA60min时拷贝数达到最高，复温再灌注45min后脑红蛋白Ngb mRNA的拷贝数较DHCA60min时略有下降，但仍高于DHCA开始时。DHCA60min时以及复温再灌注45min后脑红蛋白Ngb mRNA的拷贝数均显著高于体外循环开始前及DHCA开始时(P＜0.048)。
2、体外循环后犬血浆S-100β蛋白浓度明显升高，从循环开始至DHCA时增长幅度较高，随着DHCA的时间延长以及复温再灌注后血浆S-100β蛋白浓度仍继续升高，但增长幅度变缓。不同时间点血浆S-100β蛋白浓度间差异有显著性意义(F=1114.099，P＜0.001)。实验组和对照组对血浆S-100β蛋白浓度影响差异无显著性意义(F=0.599，P=0.461)。分组与各时间点血浆S-100β蛋白浓度无交互效应(F=0.288，P=0.678)。各时间点血浆S-100β蛋白浓度随时间变化趋势不同，从循环开始至DHCA时血浆S-100β蛋白浓度影响最大(P＜0.001)。复温再灌注45min后血浆S-100β蛋白浓度最高((?)±S=17.59±0.35 ng／ml)，但与DHCA60min时差异无显著性意义(P=0.289)。
3、体外循环后犬血浆MBP浓度缓慢升高，至DHCA60分钟，犬循环恢复开始重新灌注时，血浆内MBP浓度明显升高达最高值((?)±S=4.17±0.88 ng／ml)，在复温再灌注45分钟时血浆MBP浓度又明显下降甚至低于体外循环开始时。实验组和空白组不同时间点血浆MBP浓度有统计学差异(F=44.999，P＜0.001)，实验组与空白组间无统计学差异(F=1.988，P=0.196)，不同时间点血浆MBP浓度与分组之间无交互效应(F=1.081，P=0.376)。前二个时间点，犬血浆MBP浓度变化不大并没有明显的差异，但DHCA结束恢复脑灌注时犬血浆MBP浓度与前二个时间点有统计学差异(P＜0.001)，当脑复温灌注一段时间后，至复温再灌注45分钟时血浆MBP浓度又恢复甚至略低于体外循环开始时水平，与DHCA60分钟，犬脑刚恢复灌注时有统计学差异(P=0.001)。
4、体外循环后犬血浆TNFα浓度缓慢下降，至DHCA 60分钟，犬循环恢复开始重新灌注时，血浆内犬TNFα浓度达到实验最低值((?)±S=316.17±3.07ng／ml)，在复温再灌注45分钟时血浆TNFα浓度又缓慢上升，但没有恢复到体外循环前的水平，不同时间点间差异虽有显著性差异(F=4.258，P=0.015)，但进行不同时间点的多重比较(即两两时间比较)后发现各时间的TNFα浓度变化没有统计学差异(P＞0.152)。并且空白对照组与实验组无显著性意义(F=1.108，P=0.323)。不同时间点血浆TNF-α浓度与分组无交互效应(F=0.614，P=0.613)。
5、体外循环后犬血浆VCAM-1浓度逐渐升高，至复温再灌注45min达最大值((?)±S=35.14±3.26ng／ml)。两组间不同时间点血浆VCAM-1浓度有显著性差异(F=15.838，P=0.001)，两组间血浆VCAM-1浓度无显著性差异(F=0.525，P=0.489)。不同时间点血浆VCAM-1浓度与分组无交互效应(F=0.077，P=0.972)。在实验的前三个时间段，犬血浆VCAM-1浓度变化不大并没有明显的差异，但从DHCA60分钟至复温再灌注45分钟时段中增长幅度较高，并产生统计学差异(P=0.013)。即在犬脑部温度逐渐恢复后，外周血重新灌注后一段时间血浆中的VCAM-1浓度开始明显增高。
6、从Ngb mRNA表达与相关脑损伤指标的相关分析可得出：Ngb mRNA的表达变化与血浆S-100β蛋白在DHCA过程中存在正相关关系(偏相关系数为：0.4603，P=0.004)，相关较密切。
7、脑组织超微结构变化：
DHCA及复温再灌注后脑皮质神经元细胞超微结构改变比较明显，主要表现为线粒体肿胀明显，部分呈气球样变，线粒体嵴减少、脱落；细胞膜连续性完整，双层结构显示不清；溶酶体增生明显，并可见吞噬的线粒体。对照组与实验组脑超微结构无明显差异。
结论：
1、Hemin预处理可提高DHCA不同时期Ngb mRNA的表达，但由于拷贝数量级较大，统计学差异并不显著(F=5.153，P=0.053)。实验组与对照组相比，对改善DHCA期脑损伤的作用并不明显。氯化高铁血红素和缺氧所诱导的NGB表达存在不同机制，并且Hemin诱导的脑红蛋白mRNA表达明显慢于缺氧诱导的表达。
2、由于目前尚不能显著刺激Ngb mRNA表达，故脑红蛋白在DHCA条件下的脑保护作用并不明显，其机理及作用途径仍需进一步研究。
3、血浆S-100β蛋白、MBP、VCAM-1可作为DHCA条件下脑损伤早期的敏感指标，其中以S-100β蛋白、VCAM-1为佳，MBP次之。TNFα并不适宜作为DHCA条件下脑损伤早期的敏感指标，其在DHCA条件下脑损伤的双重作用仍需进一步探讨。
4、从Ngb mRNA表达与相关脑损伤指标的相关分析可得出，脑红蛋白有望成为DHCA条件下脑损伤早期一种新的敏感指标。其在脑损伤的诊断、预后和保护作用有待于更多的实验和临床研究。
5、脑损伤后血液中神经系统相关蛋白质时间—浓度曲线关系，综合分析多种标志物动态变化比检测单一神经系统相关蛋白质的含量变化对判断脑损伤程度、评估预后、调整治疗方案等更有临床意义。
Various techniques for the heart, brain, lung, liver, and kidney protection have been developed and with increasing cooperation between researchers from different fields, deep hypothermic circulatory arrest (DHCA) has gained more extensive clinical application. Currently, DHCA has become a very important means for surgical management of some congenital cardiac defects in infants as well as of complicated major blood vessel abnormalities in adults. Among the vital organs of the body, the brain is especially vulnerable to anoxia and ischemia. Hypothermia allows chances for the organs to evade from disastrous damages in the context of circulatory arrest by significantly lower the metabolic rate, but even so, prolonged DHCA still results in nerve system impairment of various degrees. The mechanism of DHCA-induced brain injury is complex, and most researches on brain protection following circulatory arrest give their primary attention to the means of lowering brain metabolism, which, however, does not seem to provide a solution for all the problems. A typical solution for brain protection may lie in the exploit of the mechanism of brain oxygen metabolism, so as to develop methods to promote brain oxygen preservation, transport and economic consumption. Basic studies of brain oxygen metabolismhave made great progress in recent years.
In 2000, Burmester et al discovered an oxygen-carrying globulin specific to the brain of human and mice, and gave it the name neuroglobin (Ngb), which shed light on a new scope of cerebral anoxia research. As a highly conservative single-copy gene in human, Ngb gene is located on chromosome 14q24 with a full cDNA length of 1909 bp that encodes 151 amino acids. Ngb is mainly expressed in the neuronal cytoplasm of vertebrates with high affinity to oxygen and functionally resembles hemoglobin in that it provides oxygen supply specifically to the brain and modulates the oxygen supply of the brain at the molecular level. As an important endogenous neuroprotective factor, the expression level of Ngb is positively associated with the tolerance of anoxia of the brain tissue. Ngb probably performs the functions of (1) oxygen preservation, in that it binds to oxygen in physiological conditions and release oxygen in anoxia, (2) oxygen transport, it carries oxygen through the blood-brain barrier and accelerates the diffusion of the oxygen into the mitochondria in the neurons, (3) anoxia sensors, which protects from downstream anoxic damages, and (4) clearing toxic substances such as NO.
For a long time Hemin has been used for treatment of anemia but recently, its protective role in other systems is given increasing attention. Experimental evidences have proved that Hemin can significantly enhance Ngb expression in vitro in a dose- and time-dependent manner. As a substrate and inducer of heme oxygenase (HO), Hemin can induce the expression of Ngb, resist free radical injury and reduce the toxicity of excitatory amino acids, thus provides important protection of the heart, brain, and guts against ischemic and anoxic injuries.
As techniques of molecular biology are widely applied in medical science research, increasing understanding of the pathological basis of brain injury has been obtained, and the roles of free radicals, NO, cytokines and adhesion molecules, and protein biochemical markers in extracorporeal circulation and DHCA have attracted increasing attention.
S-100β, a protein found in bovine brain by Moore in 1965 with relative molecular mass of 21×103, exists concentration-dependently in the central nervous system as an acidic calcium-binding protein. S-100βis overexpressed in the brain tissue and rapidly released into the circulation following brain injury, but due to its very short half-life, the level of this protein in the blood soon declines. Delayed functional impairment or progressive death of the glial cells following brain injury leads to S-100βleakage, and secondary brain injury causes further damage of the blood-brain barrier. As the condition worsens, secondary elevation of S-100βor persistent presence of high-level S-100βmay occur, which suggests progressive secondary brain injury.
Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons and binds tightly with myelin to maintain the integrity and functional stability of the neural sheath. In the event of neural sheath destruction, MBP can be released in the nervous system and blood circulation, and finally degraded and cleared through urination. Current data suggest that MBP is associated with acute or chronic cerebrovascular diseases, experimental allergic encephalomyelitis, multiple sclerosis and many other neurological diseases, and considered a specific biomedical indicator of organic damage of the nervous system, particularly demyelination. MBP also serves the purpose of therapeutic effect evaluation and prognostic assessment.
Tumor necrosis factorα(TNFα) is a extensively studied cytokine secreted by various types of cells. Studies suggest that this cytokine plays a double-faced role in ischemic brain injury in that TNFαworsens neuronal damage following ischemic brain injury but may also execute neuroprotective functions. But due to the insufficiency of systemic research data demonstrating the beneficial effect of TNFαin ischemic brain injury, most researchers inclined to believe that TNFαparticipates in the pathological process of brain injury as an inflammatory agent. This seems to provide new insight into therapy of ischemic brain injury.
Vascular cell adhesion molecule-1 (VCAM-1), a member of the adhesion molecule immunoglobulin superfamily, is located on the vascular endothelial cell membrane. Stimulation with inflammatory agents results in increased expression of VCAM-1 which participates in lymphocyte activation and migration and the growth and development of hemopoietic cells and is also involved in such pathological processes as inflammation and tumor metastasis. Researches have identified important roles of VCAM-1 in the inflammatory response in relation to cerebral ischemia and its progression. VCAM-1 expression can alleviate the symptoms of brain ischemia and protect against ischemic brain injury.
In this study, real-time fluorescence quantitative PCR was employed to analyze Ngb mRNA expression during extracorporeal circulation and DHCA in relation to the expressions of S-100β, MBP, TNFαand VCAM-1. The value of Ngb and other indices related to brain injury was explored in the diagnosis, therapy and prognostic assessment of brain injury due to DHCA.
Objective:
Ngb expression and plasma levels of S-100β, MBP, TNFαand VCAM-1 were detected at different time points during DHCA in dogs with or without Hemin treatment. Ultrastructural changes of the cerebral cortex was also observed to reveal the role and mechanism of Ngb and the other factors in protection against early-stage brain injury due to DHCA, thereby finding effective means for prevention and therapy of brain injury due to DHCA.
Methods:
Ten healthy hybrid dogs were randomized into control group and Hemin treatment group, and in the latter, the dogs were given 50 mg/kg Hemin 24 hours prior to the experiment. The dogs were anesthetized with 3% sodium pentobarbital followed by tracheal intubation with artificial ventilation. Cannulation of the left jugular vein was performed to collect cerebral venous blood sample, and the left carotid artery was also cannulated to measure the mean arterial blood pressure (MBP). Craniotomy was performed on the right side of the skull to obtain cerebral cortex sample. Cannulation of the aorta and the inferior and superior vena cava was performed to establish extracorporeal circulation, which was terminated till the nasopharyngeal temperature dropped to 18℃. Ninety minutes later, the circulation was restarted and terminated till a nasopharyngeal temperature of 37℃. One hour after extracorporeal circulation termination, the dogs were killed by blood depletion.
Venous blood samples were obtained from the left jugular vein immediately after extracorporeal circulation establishment, 0 and 60 minutes after extracorporeal circulation termination, and 45 minutes of reperfusion, respectively, for measurement of S-100β, MBP, TNFα, and VCAM-1 levels. Cortical samples were also obtained at these time points and frozen for real-time fluorescence quantitative PCR for Ngb mRNA measurement. At 60 minutes of reperfusion, 4% paraformaldehyde was perfused through the carotid artery and cerebral cortex samples were harvested for transmission electron microscopic observation of the ultrastructures.
The data of the measurement were statistically analyzed with SPSS11. 5 software by repeated measure analysis of variance and one-way ANOVA. Bonfferoni test was performed for paired comparison. A P value no greater than 0.05 was considered to indicate significant difference.
RESULTS
1、The establishment of extracorporeal circulation resulted in increased Ngb mRNA expression, which was especially obvious after body temperature reduction, and at 60 min after DHCA its expression reached the peak level((?)±S=565680681.56±223521679.77Copy Number/ug), followed by slight decrease after body temperature recovery. The concentration of Ngb mRNA showed significant differences between different time points (F=16.745, P＜0.001), but were comparable between the Hemin and control groups (F=5.153, P=0.392). There is no crossover effect of the concentration of Ngb mRNA between groups and different time points (F=0.249, P=0.862). At each time points of measurement, Ngb mRNA concentration exhibited different patterns of alteration, specifically, it increased after body temperature reduction till circulation arrest, reaching the peak level at 60 minutes of DHCA and a slight reduction was noted at 45 minutes of reperfusion when Ngb mRNA level was still higher than that measured at the initiation of DHCA. Ngb mRNA level at 60 minutes of DHCA and at 45 minutes of reperfusion was significantly higher than that measured before extracorporeal circulation establishment and at the start of DHCA (P＜0.048).
2、Following extracorporeal circulation establishment plasma S-100βprotein level showed significant increment, which was most obvious within the period between extracorporeal circulation initiation and DHCA. As DHCA was prolonged, S-100βprotein level kept increasing even after reperfusion, but the increment showed a gradually lowered magnitude. S-100βprotein level showed significant differences between the time points of measurement (F=1114.099, P＜0.001). But between Hemin and control groups, S-100βprotein levels showed no significant difference (F=0.599, P=0.461). There is no crossover effect of S-100βprotein level between groups and different time points (F=0.249, P=0.862). Most obvious changes of S-100βprotein level was noted in the period between extracorporeal circulation initiation and DHCA (P＜0.001). The highest S-100βprotein level occurred at 45 minutes of reperfusion((?)±S=17.59±0.35 ng/ml), but this high level showed no significant difference from that measured at 60 minutes of DHCA (P=0.289).
3、Plasma MBP level increased gradually following extracorporeal circulation initiation till 60 minutes of DHCA, and at reperfusion, plasma BMA reached its peak level ((?)±S=4.17±0.88 ng/ml), but at 45 minutes of reperfusion, MBP level underwent a significant reduction even to a level below that before extracorporeal circulation initiation. MBP level showed significant differences between the time points of measurement (F=44.999, P＜0.001). But between Hemin and control groups, MBP level showed no significant difference (F=1.988, P=0.196). There is no crossover effect of MBP level between groups and different time points (F=1.081, P=0.376). Between extracorporeal circulation initiation and 60 minutes of DHCA, plasma DHCA level did not undergo significant changes until the termination of DHCA and initiation of reperfusion (P＜0.001), but at 45 minutes of reperfusion, BMP level recovered and became even lower than the level before extracorporeal circulation, showing significant difference from the level at 60 minutes of DHCA (P=0.001).
4、Plasma TNFαlevel was slowly reduced after extracorporeal circulation initiation till 60 minutes of DHCA, and at the time of reperfusion, TNFαlevel reached its lowest ((?)±S=316.17±3.07 ng/ml), followed by slow increase at 45 minutes of reperfusion but failed to recover its former level. Its level showed significant differences between the time points (F=4.258, P=0.015), but these differences were not supported statistically after multiple comparison (P＞0.152). TNFαlevel was not significantly different between the control and Hemin groups (F=1.108, P=0.323). There is no crossover effect of TNFαlevel between groups and different time points (F=0.614, P=0.613).
5、Plasma VCAM-1 level increased gradually following extracorporeal circulation initiation and at 45 minutes of reperfusion, plasma VCAM-1 level reached its peak level ((?)±S=35.14±3.26ng/ml). Plasma VCAM-1 level showed significant differences between the time points of measurement (F=15.838, P=0.001). But between Hemin and control groups, Plasma VCAM-1 level showed no significant difference (F=0.525, P=0.489). There is no crossover effect of Plasma VCAM-1 level between groups and different time points (F=0.077, P=0.972). In the former 3 stages of the experiment, VCAM-1 level did not undergo significant alterations, but from 60 minutes of DHCA to 45 minutes of reperfusion, VCAM-1 level showed obvious increase (P=0.013). In other words, VCAM-1 level began to increase obviously following reperfusion after brain temperature recovery.
6、Correlation analyses between Ngb mRNA expression and the brain injury indices described above revealed that Ngb mRNA expression was in close positive correlation with plasma S-100βlevel during DHCA (with coefficient of partial correlation of 0.4603, P=0.004).
7、After DHCA and reperfusion, obvious changes were observed in the ultrastructure of the cortical neurons, including swelling of the mitochondria and reduction of the mitochondrial crest. The cell membrane remained continuous and intact with clear bilayer structure. Lysosome proliferation was obvious, and phagocytosed mitochondria could be seen. These changes were not significantly different between the control and Hemin groups.
CONCLUSION
Hemin pretreatment may enhance Ngb mRNA expression at different phases of DHCA, but due to the large magnitude of the mRNA copies, no statistically significant differences are produced (F=5.153, P=0.053). Hemin failed to show obvious effect in improving brain injury during DHCA in comparison with the control group. Hemin and anoxia induce Ngb expression through different mechanisms, and the former seems to induce Ngb expression at a much lower pace than the latter.
As currently no effective means are available to sufficiently stimulate Ngb expression, the protective effect of Ngb against brain injury is not obvious during DHCA, and the mechanism needs further exploration.
Plasma S-100βprotein, MBP, and VCAM-1 can be sensitive indicators of early brain injury during DHCA, but S-100βprotein and VCAM-1 serve this purpose better than MBP. TNFαdoes not seem to suit for this purpose, and its double effects in brain injury in relation to DHCA needs further investigation.
Ngb has the potential as a novel sensitive indicator for brain injury during DHCA, but its value in the diagnosis, prognostic evaluation and protective effect in brain injury awaits more extensive laboratory and clinical research.
Analysis of the time-concentration curve of the nervous system-related proteins in the blood following brain injury and comprehensive evaluation of the dynamic changes of multiple markers, other than single such proteins, can be more valuable for brain injury severity assessment, prognostic evaluation and treatment planning.
引文
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[3] 赵贇，胡克俭，程玥，等．犬的体外循环模型建立和管理[J]．中国体外循环杂志，2004；2(4)：217-218．
[4] 上海第二医学院，北京儿童医院主编．小儿内科学[M]．第二版人民卫生出版社，1980：288．
[5] 杨运海，罗海燕，谢艾妮，等．无血预充兔体外循环模型的建立[J]．中国比较医学杂志，2005；15(6)：372-374．
[6] Kim WG, Moon HJ, Won TH, et al. Rabbit model of cardiopulmonary bypass [J]. Perfusion, 1999; 14(2):101-105.
[7] 田鑫，江基尧，邱永明，等．大鼠深低温停循环复苏模型的建立[J]．中华神经外科杂志，2004；20(5)：417-420．
[8] Jungwirth B, Mackensen GB, Blobner M, et al. Neurologic outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats: description of a new model[J]. J Thorac Cardiovasc Surg, 2006; 131(4):805-12.
[9] Burmester T, WeichB, Reinhardt S, et al. A vertebrate globin expressed in the brain. Nature, 2000,407 (6803) :520～523
[10] Sun Y J, Jin K L , Greenberg D A , et al. Neuroglobin is up —regulated by and protects neurons from hypoxic—ischemic injury. Proc Natl Acad Sci U S A ,2001 ,98 (26) :15306～15311
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