【作者】 刘育杰；

【导师】 王爱民；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 随着交通业、工矿业的发展,交通事故、高处坠落、重物砸伤等高能量致伤因素导致的脊髓损伤发病率逐年增高,并表现出多发伤发生率高、伤情复杂、伤情重、高死亡率及伤残率等特点。经过文献检索发现,关于“以脊柱脊髓损伤为主要创伤的严重多发伤”的研究报道很少,相关基础研究缺乏。前期研究中,我们首次将此类型多发伤命名为“脊柱脊髓型严重多发伤”,并将其定义为:在严重多发性损伤中(按简明损伤定级-损伤严重度评分(AIS-ISS)系统评分,ISS>17分),以脊柱脊髓损伤为主,其AIS≥4分,ISS≥16分。同时发现,此类多发伤患者急性期常出现血流动力学紊乱,伤后急性期血流动力学的稳定与否直接关系到救治成功率。但对于脊柱脊髓型严重多发伤伤后急性期血流动力学变化规律和特点尚不清楚。同时,为纠正此类创伤造成的休克,临床一般性救治原则是补足血容量,尽早使用缩血管药物。因而,此阶段心血管对缩血管药物的反应性(简称心血管反应性,cardiovascular reactivity)直接关乎救治结果,但其变化规律亦未见相关研究报道。鉴于脊柱脊髓型严重多发伤发病率日渐增高,因此,积极开展脊柱脊髓型严重多发伤早期血流动力学变化病理生理学相关研究,改进治疗手段,对于改善预后,提高救治成功率,降低死亡率和伤残率均具有重要意义。主要方法:由于此类创伤病例收集难度大,同时患者入院时常因伤情重笃,为救治常施加各种干预因素,很难开展相关的采样和药物治疗等临床研究,因此,首先我们以高位脊髓损伤(C7-T1)、骨折(单侧胫骨骨折)、失血为主要致伤因子,建立了脊柱脊髓型严重多发伤大鼠模型;其次,在此模型基础上,利用在体血流动力学检测系统观察了伤后急性期(伤后1-6 h)血流动力学变化规律,并借助彩色微球技术,在微创条件下在体连续的观察了各重要脏器器官血流量变化特点,同时利用酶联免疫吸附实验(ELISA)和放射免疫测定法(RIA)检测了血中三种主要交感缩血管神经递质(去甲肾上腺素、肾上腺素、神经肽Y)变化;再次,为研究伤后心血管反应性变化规律,我们在此模型上先通过检测去甲肾上腺素(NE)升压反应从而评价了心血管总反应性变化特点,继而以内脏血管-肠系膜上动脉(SMA)为研究对象,利用微循环图像计算机分析系统和离体血管环张力测定系统分别从在体和离体两个方面测定了血管反应性变化,并通过血流动力学检测系统在体观察了心脏反应性变化。最后,为初步探讨血流动力学及心脏反应性变化机制,我们利用放射受体配基结合分析(RBA法)观察了伤后急性期左心室心肌细胞膜NE主要作用受体α1-肾上腺素能受体(AR)、β-AR数量及其亲和力变化,并借助非标记底物法和RIA研究了介导NE正性肌力作用、正性传导作用、正性变时作用的主要信号通路-β-AR-腺苷酸环化酶(AC)系统变化。主要结果:①我们建立的脊柱脊髓型严重多发伤大鼠模型在伤后急性期即可出现血流动力学紊乱、酸碱失衡、治疗效果差等重症休克表现,和单发伤对比存在多发伤相互加重效应,符合此病早期临床特征;②脊柱脊髓型严重多发伤大鼠平均动脉压(MAP)在伤后1～6 h,心率(HR)在伤后0、0.5、4、5、6 h,左室收缩压(LVSP)在伤后3～6 h,左心室内压最大变化速率(±dp/dtmax)、总外周血管阻力(TPR)、心输出量(CO)在伤后0.5～6 h显著降低。上述指标和其它组相比,差异均存在统计学意义(P<0.05或P<0.01);③脊柱脊髓型严重多发伤大鼠血清肾上腺素(EPI)浓度在伤后0.5、1、2、3、5 h,NE浓度在伤后2、3、6 h轻度高于伤前(P<0.05),神经肽Y(NPY)浓度在伤后0.5～2 h和5～6 h呈小幅双峰增长(和伤前比,P<0.05);伤后各时间点血NE、EPI、NPY浓度均远低于创伤失血组大鼠(P<0.01);④脊柱脊髓型严重多发伤大鼠伤后各时间点各器官血流量均显著降低(和假手术组比,P<0.05),但和创伤失血组大鼠不同的是,伤后各时间点心、脑局部血流量降低幅度和其它器官血流量比无明显差异;⑤脊柱脊髓型严重多发伤大鼠NE升压反应在伤后1～6 h均显著增高(和假手术组及创伤失血组比,P<0.01)。SMA反应性呈现双相变化:休克1～3 h,NE对SMA最大收缩力(Emax)和半数有效浓度(pD2(-log[EC50]))均显著高于假手术组(P<0.05或P<0.01),量效曲线左移。伤后4～6 h,SMA对NE的Emax、pD2显著低于假手术组(P<0.05或P<0.01),量效曲线右移;⑥脊柱脊髓型严重多发伤大鼠伤后1-6 h,给予NE后心功能各项指标(LVSP、±dp/dtmax)增幅始终显著高于伤前和创伤失血组(P<0.05或P<0.01);⑦伤后1h,左心室心肌细胞膜α1-AR和β-AR受体最大结合数目(Bmax)达到最高点,其中,β-AR增加幅度最为显著,至伤后6h,β-AR Bmax依然维持在较高水平(和伤前比,P<0.01)。两种受体Kd(the equilibrium dissociation constants)在伤后1h、3h变化不大,伤后6h则出现明显增高(P<0.01),提示受体亲和力下降。伤后心肌组织AC基础活性及cAMP浓度随着时间延长逐渐降低,二者呈正相关(相关系数r=0.9392,p<0.05)。异丙肾上腺素(ISO)激活AC活性在伤后各时间点均明显最高(P<0.01),提示β-AR-G蛋白-AC-cAMP信号系统在伤后6h内的反应性均增强。从增高程度看,伤后1h高于正常值2.5倍,随着受伤时间的延长,增高程度逐渐下降。法司可林(FSK)激活的AC活性在伤后各时间点变化不大,提示AC催化亚基蛋白自身无显著改变。氟化钠(NaF)激活的AC活性在伤后1 h变化不大,伤后3、6 h,则显著降低(和伤前比,P<0.01),提示伤后3、6h,G蛋白和AC催化亚基出现脱偶联改变。结论:①本实验建立的脊柱脊髓型严重多发伤大鼠模型能较好地模拟脊柱脊髓型严重多发伤伤后早期病理生理学变化,能够满足本研究的需要,且具有伤情稳定、操作简便、重复性好等特点;②脊柱脊髓型严重多发伤大鼠伤后急性期血流动力学紊乱出现时间早,持续时间长,程度重,无代偿性增高。各重要脏器器官血流量伤后早期即出现显著降低,未出现代偿性器官血流量重新分布。血中交感缩血管神经递质相对缺乏。血流动力学恶化程度和创伤严重程度呈正比;③和创伤失血性休克不同的是,脊柱脊髓型严重多发伤大鼠心血管总反应性在伤后1～6 h均显著增高,但此阶段血管反应性呈现双相变化,而心脏反应性在伤后1～6 h均显著增高。从时相点及增幅看,心脏反应性增高可能是导致伤后心血管总反应性增高的主要原因;④脊柱脊髓型严重多发伤大鼠伤后急性期一方面出现“血交感缩血管神经递质相对不足”、“AC基础活性降低继发心肌组织cAMP含量下降”、“G蛋白和AC催化亚基脱偶联”等改变,导致伤后出现严重的心功能下降及血流动力学恶化;同时,也出现心肌细胞胞膜AR数目增多(以β-AR最为明显),导致心脏反应性增加,这可能是机体发挥代偿作用的重要机制之一。更多还原

【Abstract】 With the development of industry and transportation, the cause of spinal cord injury (SCI) are always associated with high energy incidents such as traffic accident, aloft fall, crush or crash by heavy objects. Multiple traumas occur quaque, and the traumatic conditions always are complex and critical. After system literature retrieval, little researches were found about multiple trauma in which SCI was leading cause of trauma. In our formal study, this kind of trauma were named of“multiple trauma headed by SCI”firstly and be defined as“a kind of serious multiple trauma (ISS>17) in which SCI was the main trauma (AIS≥4, ISS≥16)”. At the same time, we found that hemodynamic disorder always occur during acute stage and have great influence on the curative results. Stabilizing hemodynamics is the main goal during emergency medical treatment. But little was known about the change mode of hemodynamics of multiple trauma headed by SCI. This kind of multiple trauma always lead to severe shock whose basic clinical remedy rules are“supplement blood volume and use vaso-excitor drugs early”. Therefore, cardiovascular reactivity to vaso-excitor (cardiovascular reactivity) is crucial to therapeutic result. However, cardiovascular reactivity is still unknown in emergency treatment during acute stage about this kind of trauma. For the above reasons, the hemodynamic and cardiovascular reactivity researches about multiple trauma headed by SCI are needed urgently to improve clinical therapeutic results and decline the mortality and invalidity rate.Methods: Firstly, as it is hard to collect cases and to make experiment on those patients for the crisis pathogenetic conditions, a new rat model of experimental multiple trauma headed by SCI had been established according to the main cause factors of this kind of trauma: high level SCI (T1-C7), fracture (lateral tibia) and bleeding. Secondly, the change patterns of hemodynamics were observed by hemodynamic monitor system on this model during acute stage. Catecholamine and neuropeptide Y (NPY) in blood, the main sympathesis vasoconstrictor neurotransmitter, were measured by ELSA or RIA. Organs regional blood flow (RBF) were monitored consecutively by color-labeled microspheres technique in vivo, and total peripheral resistance (TPR) rise and cardiac output (CO) were calculated simultaneously. Thirdly, to investigate cardiovascular reactivity, total cardiovascular reactivity was measured by the pressor effect of norepinephrine (NE) at different time after trauma. Then the vascular reactivity was measured separately by microcirculation monitor system in vivo and by vascular ring tension system in vitro. The cardia reactivity was measured by monitor the heart function after injected NE (3ug/kg) in vivo. The main receptors of NE are beta (β) - and alfa1 (α1)-adenoreceptor (AR) in heart and link to AR-adenylate cyclase (AC)-cAMP signal system. Any changes of reaction to NE of heart must original to this signal system. For this reason, we checked the numbers and affinity ofβ-AR andα1-AR by radioligand binding assay (RBA) of receptors and AC activity by unlabeled substrate assay.Results:①This animal model exhibited typical characters of multiple trauma headed by SCI that are include abnormal of hemodynamics, blood gases in early period and high mortality even after resuscitation.②MAP of multiple trauma headed by SCI rats in 1-6 h, HR in 0, 0.5, 4, 5, 6 h, LVSP in 3-6 h,±dp/dtmax , CO and TPR in 0.5-6 h drop dramatically compared with other groups (P<0.05 or 0.01).③The concentration of epinephrine (EPI) in 0.5, 1, 2, 3, 5 h, NE in 2, 3, 6 h rise slightly compared with sham (P<0.05). The concentration of NPY exhibited two peaks compare with normal: the first rise emerges between 0.5-2 h and the second between 5-6 h after trauma (compared with normal, P<0.05). The concentrations of NE, EPI, NPY are far lower than trauma-hemorrhage rats (P<0.05).④Organ RBF drop significantly and simultaneously which did not have significant difference with other organs (P>0.05).⑤The pressor effect of NE in this rats model was significantly higher than sham and trauma-hemorrhage rats at all the time point (P<0.01). The vascular reactivity of SMA to NE was increase significantly 1-3 h after trauma as compared with sham: the Emax and pD2 were significantly increased (P<0.01), the cumulative dose-response curves were shift to the left. The aboved indexes decrease significantly at 4-6 h after trauma and the cumulative dose-response curve were shift to the right.⑥The cardia function indexes of multiple trauma rats rise significantly after i.v. NE in vivo whereas decreased in trauma-hemorrhage rats during 1-6h after trauma(P<0.01).⑦ Bothα1- andβ- AR density reach the summit 1h after trauma (P<0.01) and decrease gradually as the time went on.β-AR density increase more significantly thanα1-AR or sham(P<0.01). It still keep higher level until 6h after trauma. No significant difference in the equilibrium dissociation constants (Kd) for both receptors was observed during 1-3 h and rise simultaneously 6h after trauma. Both basal AC activity and the concentration of cAMP in membrane preparations drop dramatically after trauma and their relationship was significant correlation (r=0.9392,P<0.05). AC activity stimulated by isoproterenol increased during 1-6 h after trauma and the highest rise at 1 h after trauma (by 2.5 times higher over the sham). It implies hyper-reactivity ofβ-AR-G protein-AC-cAMP signal transduction system which persisted 6h after trauma. No significant statistic difference was observed on AC activity stimulated by forskolin. It implies that there are no change on AC catalytic subunit protein itself. AC activity stimulated by NaF showed little increase during 1 h after trauma and a dramatic decreasing appeared during 3-6 h after trauma (P<0.01). It implies uncoupling of G protein and AC catalytic subunit during this period of time.Conclusions:①This animal model with trauma of stable degree can be repeated easily and fit for the needs of early pathophysiological study of multiple trauma headed by SCI.②Appearance early, long duration, serious degree and compensation handicap are the characters of hemodynamics and heart function disorder of multiple trauma rats headed by SCI. Organ RBF drop too and did not exhibit redistribution unlike trauma-hemorrhage rats. The deterioration of hemodynamics is positive correlation with the degree of trauma. The concentration of sympathesis vasoconstrictor neurotransmitter show relative deficiency.③The total cardiovascular reactivity rise (hypereactivity) and the vascualar reactivity exhibited biphasic change during 1-6 h after trauma. But the cardia reactivity exhibited hyper-reactive during 1-6 h. With reference to the change time points, tendency and degree, the increased cardia reactivity could be one of the main reasons for the cardiovascular hypereactivity but vascular reactivity.④Multiple trauma rats headed by SCI exhibited high reactivity of AR-AC-cAMP signal transduction system that mainly due to increased density of AR after trauma during acute stage. That could be one of the important compensation mechanisms of cardia hypersensitivity after trauma. At the same time, some changes, such as relative deficency of sympathesis vasoconstrictor neurotransmitter, low activity of basal AC activity and the concentration of cAMP in myocardium, uncoupling G protein and AC catalytic subunit, also occur and could be one of the main reasons of aggravation of heart function and hemodynamics after trauma.更多还原