【作者】 徐翔；

【导师】 古妙宁；

【作者基本信息】 南方医科大学 ， 麻醉学， 2008， 硕士

【摘要】 颅脑外伤在平时和战时都是最严重的创伤之一,合并失血性休克的重度脑外伤死亡率极高。休克时常输注大量液体以维持血压稳定,却往往造成脑组织水肿,引起继发性损伤;缺血/再灌注时产生大量的氧自由基,常造成脑细胞的不可逆损害;而常规应用脱水利尿药治疗脑水肿降低颅内压（intracranial pressure,ICP）时,又会导致有效血容量减少,加重休克。近年来,失血性休克合并颅脑外伤早期的液体治疗和容量管理越来越受重视,小容量高渗盐溶液及其复合溶液复苏失血性休克的作用已经在动物实验中得到证实,并在临床上有良好的治疗作用。在动物实验中,不少学者发现高渗盐溶液有降低颅内压的作用,但在临床上尚未得到确切证据。既往文献中氯化钠的浓度大多高于7.5%,对浓度低于7.5%的高渗盐的研究鲜有报导;且文献中多为单纯高渗氯化钠溶液,对添加了胶体的高渗盐复合液对颅内高压合并休克的作用报道也较少。本实验主要观察高渗氯化钠羟乙基淀粉注射液（hypertonic sodium chloridehydroxyethyl starch 40 iniection,HSH）复苏休克降低颅内压作用,通过检测脑组织丙二醛（malondialdehyde,MDA）含量、超氧化物歧化酶（superoxide dismutase,SOD）活力,探讨HSH对脑组织缺血/再灌注损伤主要环节的影响,同时观察脑组织相应形态学的改变,以评估HSH在脑保护方面的应用价值。材料与方法1、动物与分组健康杂种犬30只,雌雄不拘,体重10kg～20kg,由南方医科大学附属南方医院实验动物中心提供。随机分为羟乙基淀粉溶液组（HES组）,乳酸盐林格液组（RL组）,7.5%氯化钠溶液组（HS组）和高渗氯化钠羟乙基淀粉注射液组（HSH组）,其中HSH组按剂量不同分为HSH4组、HSH8组和HSH12组。每组5只动物。2、动物模型的建立及复苏外周静脉穿刺置管,静注3%戊巴比妥钠溶液（1ml/kg）诱导麻醉。麻醉平稳后仰卧固定于操作台上,经口插入9号气管导管,连接麻醉机,固定,调节呼吸参数（呼吸频率15/min～30/min,潮气量250ml～430ml）使ETCO₂维持于（32～37）mmHg。静脉持续泵入维库溴铵(0.08mg·kg^-1·h^-1)、吸入1%异氟烷和纯氧维持麻醉。行右颈静脉切开,插入18G静脉导管,连接监测仪,监测中心静脉压（CVP）。左股动脉切开,插入20G肝素化套管,监测动脉血压。犬头顶部去毛,行皮肤正中纵行切口切开头皮,显露顶骨。于左顶骨钻孔,直径6mm～10mm,将可注水球囊置入并送至硬膜外腔,固定球囊,缝合头皮。右侧顶骨钻孔作脑室插管,脑脊液充满塑料管后固定并接L型测压管测脑室内压,调节零点与左外耳道平行。两侧颅骨钻孔缺损均用骨蜡封闭,保证测压的可靠性。向可注水球囊内注水,使脑室内压比基础压力升高10mmHg并维持。右股动脉穿刺置管,15min内匀速放血,使MAP降低到40mmHg,并调整失血量使血压维持此水平,持续60min完成急性颅内高压伴失血性休克模型制作,随后进行复苏。HSH4组按4ml/kg,HSH8组按8ml/kg,HSH12组按12ml/kg,HES组按等倍失血量,RL组按三倍失血量,HS组按6ml/kg输入,所有液体均于20min内输完。于复苏4h开颅取颞叶全层脑组织,一部分用于测定MDA含量及SOD活力,另一部分用于形态学研究。3、观察指标3.1复苏过程连续监测MAP、CVP、HR、ICP。3.2分别于实验前（T1）、造模后（T2）、复苏后20min（T3）、复苏后40min（T4）、复苏后1h（T5）、复苏后2h（T6）、复苏后3h（T7）、复苏后4h（T8）共8个时点记录数据:MAP、ICP。3.3 MDA含量及SOD活力测定:于复苏4h打开颅骨,在颞叶切取一约1cm³的脑组织块,用于测定MDA含量及SOD活力。MDA含量测定采用硫代巴比妥酸法,SOD活力测定采用黄嘌呤氧化酶法。试剂盒均由南京建成生物工程研究所提供,测定方法按试剂盒说明书。3.4病理组织学研究:脑组织置于4%甲醛溶液中固定24h,乙醇梯度脱水,常规石蜡包埋切片,HE染色,封片后显微镜下观察及摄片。3.5脑组织超微结构检查:取大脑皮层修成1mm³的小块,常规方法制备标本后行透射电镜检查。4、统计学处理实验数据以均数±标准差（（?）±s）表示,先行方差齐性检验,每组动物平均体重,球囊注水量,放血量,基础颅内压,注水后颅内压及各组MDA、SOD等指标采用单因素方差分析进行处理,各个时点的MAP、ICP组内比较、组间比较、两两比较和多重比较采用重复测量数据的方差分析。以P＜0.05为差异有统计学意义。统计学检验的计算采用SPSS13.0统计软件。结果1、动物体重、复制急性颅内高压伴失血性休克模型的球囊注水量、放血量、基础颅内压以及注水后颅内压各组差异均无统计学意义（P＞0.05）。2、各组复苏前后MAP、ICP变化造模前及复苏前各组动物MAP相似,无统计学意义（P＞0.05）。复苏后各组液体均能有效提高MAP（与复苏前比较F=133.096,P＜0.001）,组间差异没有统计学意义（P＞0.05）,但各组反应不同。HSH提升血压速度最快,在复苏20min时就能恢复到初始水平。HS组在复苏2h后血压开始下降,至实验结束时降至休克水平,其余各组血压均能维持至实验结束,组间无显著差异（P＞0.05）;时间与复苏液体之间存在交互效应（F=2.727,P=0.001）。复苏前各组动物ICP相似,无统计学意义（P＞0.05）。复苏后各组ICP变化有显著差异（F=133.096,P＜0.001）:HS组、HSH4组、HSH8组和HSH12组的ICP迅速降低,但HSH4组维持时间短,2h后缓慢上升。HS组ICP于复苏40min降至最低值后逐渐上升,但至复苏后4h仍低于基础值。HSH8组1h后下降至最低值,接近基础值水平,并维持4h。组间对ICP影响有显著差异（F=62.678,P＜0.001）,从各时间点看,HSH8、HSH12与HS组降低ICP差异无显著性（P＞0.05）,均强于HSH4组（P＜0.01）:时间和复苏液体之间存在交互效应（F=48.592,P＜0.001）。复苏后RL组和HES组ICP都呈上升趋势。3、各组脑组织MDA含量、SOD活力复苏4h,HES组、HS组、HSH组脑组织的MDA含量均较RL组明显降低,SOD活力明显升高。其中HSH8组及HSH12组改变较其他组明显,与HS组、RL组和HES组比较均有统计学意义（P＜0.05）。4、脑组织病理学检查显微镜下,HS组神经元细胞及胶质细胞体积缩小,核深染,细胞与周围组织间隙增大。HES组及RL组均显示脑皮质水肿,神经元和胶质细胞严重水肿,空泡变性,细胞核溶解消失,小血管扩张、渗出,局部组织溶解。HSH4组脑组织结构的破坏改善,表现为水肿减轻,仅少量神经元轻度水肿,渗出减少,且无坏死灶发现。HSH8组脑组织结构的破坏进一步减少,水肿进一步减轻,神经元无明显水肿,轴突形态清晰,无明显渗出及坏死灶。HSH12组脑组织结构变化与HSH8组接近,无明显水肿,神经元及胶质细胞形态基本正常,小血管无充血,无坏死灶发现。5、大脑皮层超微结构检查HES组:少量神经元及胶质细胞的形态不完整,细胞核肿胀明显,染色质分布稀疏;可见线粒体肿胀及脱颗粒现象,高尔基体肿胀明显。HS组:部分神经元及胶质细胞的形态不完整,细胞周围有较大间隙,染色质边集较明显;线粒体数量减少,胞浆内可见空泡化;毛细血管周围间隙明显。RL组:电子密度降低,神经元及胶质细胞肿胀明显,部分神经元及胶质细胞包膜破裂,细胞器消失。细胞核明显肿胀,线粒体肿胀,嵴消失,并有脱颗粒现象;高尔基体肿胀,数量减少。HSH组:HSH4组的神经元及胶质细胞形态基本完整,细胞核清晰可见;线粒体轻度肿胀,排列规则,粗面内质网形态正常;毛细血管结构基本正常。HSH8组及HSH12组神经元及胶质细胞形态基本完整,胞核清晰,核仁明显;可见较为丰富的粗面内质网和线粒体;线粒体排列规则;毛细血管结构正常。结论1、高渗氯化钠羟乙基淀粉注射液可有效复苏犬急性失血性休克。2、高渗氯化钠羟乙基淀粉注射液可显著降低犬创伤性颅内高压。3、高渗氯化钠羟乙基淀粉注射液可显著降低缺血/再灌注时MDA的生成,提高SOD活力。4、从病理学检查及超微结构看,高渗氯化钠羟乙基淀粉注射液可减轻急性颅内高压合并失血性休克犬的脑组织水肿情况,降低细胞的损伤程度。更多还原

【Abstract】 Craniocerebral trauma, one of the most serious traumas in clinical, can cause a high mortality when associated with hemorrhagic shock. In hemorrhagic shock patients, application of volume therapy to maintain blood pressure may cause cerebral edema. Subsequently, a great deal of oxygen free radicals produced in ischemic-reperfusion could produce irreversible damage of brain cells. In the other hand, application of dehydrate and diuretic to treat cerebral edema and decrease intracranial pressure would reduce effective blood volume and aggravate hemorrhagic shock.Recently, an increasing attention has been paid on volume therapy and management during early stage of hemorrhagic shock associated with craniocerebral trauma. Some studies have confirmed the resuscitation effect of small capacity hypertonic saline solution and its combined solutions through animal experiment and clinical trial. Although investigators have found hypertonic saline solution can reduce ICP through animal experiment, there is no solid proof in clinical practice. But hypertonic saline, in which concentration of NaCl is less than 7.5%, is reported rarely.In this study, we designed a dog acute intracranial hypertension and hemorrhagic shock model to observe the effects of hypertonic sodium chloride hydroxyethyl starch 40 injection （HSH） on circulating blood volume, ICP and cerebral edema and oxygen free radicals. Furthermore, we evaluated the application value of HSH in cerebral protection by detecting the contents of malondialdehyde （MDA）, the activity of superoxide dismutase （SOD） and the change of cerebral tissues.Materials and methods1. Animals and groupsThirty adult mongrel healthy dogs, male or female, weighing 10-20 kg, were provided by Laboratory Animal Center of Southern Medical University. All the animal were randomly divided into 6 groups, they are group HES, group RL, group HS and group HSH4, group HSH8, group HSH12. Each group consisted of 5 dogs.2. Model and resuscitationAnesthesia was induced with venous injection of 3% pentobarbital （1 ml/kg）, followed by endotracheal intubation and mechanical ventilation. Adjusted respiratory parameters （RR=15bpm-30 bpm, Vt =250ml-430ml） to keep ETCO₂ changing between 32-37 mmHg. Intravenous infusion of vecuronium, Inhalation of 1% Isoflurane and oxygen maintains anesthesia. Incise right jugular vein and insert catheter （18 G） to monitor CVP. Left femoral artery incision was for monitoring arterial blood pressure. Indwell catheter after cystostomy. A catheter with balloon was placed in epidural cavity through an 10mm burr hole at the parietal bone and then fixed. Another catheter was placed in right parietal lobe through a 2-mm burr hole at the parietal bone and then connected to a piezometric catheter for monitoring ICP. Adjust zero point to parallel with left external auditory canal. Water was inject into the balloon, making the ICP 10 mmHg higher than the basic pressure and maintain the pressure. Kept MAP at near 40 mmHg by bloodletting through right femoral artery in 15 minutes and maintain for 60 minutes. Thus the model of acute intracranial hypertension and hemorrhagic shock was accomplished. Then resuscitation followed. All the dogs were resuscitated with either Ringer-Lactates solution （RL, three times as the amount of blood loss）, hydroxyethyl starch （HES, equivalent to the amount of blood loss）, 7.5 %NaCl （HS, 6 ml/kg）, HSH （4 ml/kg, 8 ml/kg, 12 ml/kg）. All kinds of liquid were infused in 20 minutes. After resuscitation for 4 hours, brain tissues of temporal lobe were taken for detecting MDA and SOD, and morphologic observation.3. Observation indexes3.1 Mean arterial pressure, cerebral perfusion pressure, intracranial pressure, central vein pressure were monitored consecutively.3.2 Data of MAP and ICP was record at baseline （T1）, after making model （T2）, 20 minutes after resuscitation （T3）, 40 minutes after resuscitation （T4）, 1 hour after resuscitation （T5）, 2 hours after resuscitation （T6）, 3 hours after resuscitation （T7）, and 4 hours after resuscitation （T8）.3.3 Detection of contents of MDA and activity of SOD: After resuscitation of 4 hours, the cranium was opened and brain tissue was removed for detecting. Both of the kits were provided by Nanjing Jiancheng Biology Engineering Institute. Contents of MDA and activity of SOD were measured based on the test kit instruction.3.4 Histopathological study: The brain tissues were removed and fixed in a 4% Formalin solution for 24 hours, desiccated by ethanol and embedded in paraffin. The histopathological changes of brain were observed by microscope according to HE dyeing.3.5 Ultrastructural examination of brain tissue: Cut the brain tissue of cerebral cortex into small pieces of 1mm³, for sample preparation and then electron microscope examination.4. Statistical analysis Results were expressed as mean±standard deviation （（x|-）±s）. First, Test of homogeneity of variances was analyzed. Then one-way analysis of variance （ANONA） was used to evaluate the basic status data and contents of oxygen free radicals, and analysis of variance of repeated measure data was used for comparisons between groups. Post Hoc multiple comparisons were analyzed by using SNK test. SPSS 13.0 were used to analyze the data. Differences were accepted as statistically significant when P values were less than 0.05.Result1. In mean weight of dogs, size of water balloon, volume of blood loss, baseline of intracranial pressure, intracranial pressure after balloon inflation. There was no statistical significance among the groups （P>0.05）.2. Changes of MAP and ICP after resuscitationMAP increased markedly after resuscitation in all groups（compare with MAP before resuscitation , F= 133.096, .P<0.001）. There was no statistic significance among the groups （P> 0.05）. But it responded most fast in group HSH and reached a peak in 20 minutes after the recovery. MAP decreased remarkably after two hours in group HS （P <0.01）, but the other three groups could maintain until the end of experiment. There was interaction-effect between time and liquid（F= 2.727, P= 0.001）.There was no statistical significance in ICP among groups before resuscitation （P>0.05）. After resuscitation, there was statistical significance in each group（F= 133.096, P<0.001）. ICP decreased significantly in group HS, group HSH4, group HSH8 and group HSH12, but increased after 2 hours in group HSH4. ICP in group HS maintained the level of less than nomal. ICP increased significantly after resuscitation in group RL and group HES.3. MDA, SOD After resuscitation for 4 hours, MDA contents of brain tissue in group HSH, group HS and group HSH was significantly lower than that in group RL, associated with increase of SOD activity（P<0.05）. Significant changes were observed in group HSH8 and group HSH12, and there is statistical significance when compared with group HS, group RL and group HES.4. Histopathology of brain tissueLight microscopic findings in group HS included reduced size of cells, dark-dyeing nuclear and enlarged space around the cells. Brain tissue of group HES and group RL swelled severely and local constitution lysis appeared. HSH improves the structure of brain tissue, revealed by less edema and exudation and no necrosis was found.5. Changes of ultra-structure in palliumObserving pallium in group HES by transmission electron microscope: The integrity of some neuron was damaged, mitochondrion was swelling and vacuolization, Golgi body was swelling obviously.The integrity of several neuron was damaged in group HS. Vacuolization appeared intracytoplasm. Other changes were apparent gaps among cells, apparently edged chromatin and less mitochondrion.In group RL, neuron was swelling obviously, some of the cell envelope damaged and the cell organ disappeared. The cell nucleus and Golgi body were swelling. Mitochondrion swelled, cristae disappeared and degranulated.In group HSH, the damage became less, which mainly laid in that form of neuron and nuclei was almost normal, chromatin was mainly composed of euchromatin; swelling and vacuolization of mitochondrion became less, and Golgi body was less swelling.Conclusion 1. HSH can resuscitate hemorrhagic shock effectively.2. HSH can reduce ICP of intracranial hypertension after traumatic brain injury in dog.3. HSH can reduce oxygen free radicals levels in brain tissue and relieve tissue ischemia-reperfusion injury.4. According histopathological study and ultrastructural examination, HSH can relieve edema of brain tissue in acute intracranial hypertension and hemorrhagic shock of dog.更多还原