超声联合微泡对肝断裂伤止血作用的实验研究

发布时间：2018-05-16 03:24

本文选题：造影剂微泡 + 超声空化效应　；参考：《广州医科大学》2014年硕士论文

【摘要】：背景肝脏组织结构脆弱，血流十分丰富，且其体积较大，位置比较固定，因此受到暴力打击时容易发生损伤破裂。应对肝破裂的急救，临床上主要采用外科手术治疗，包括纱布块填塞法、电凝止血法、氩气止血法、单纯缝合修补、肝动脉栓塞或结扎术、肝部分切除术等。虽然效果显著，但是对患者有较大的手术损伤，而且，手术治疗常需要一定的设备及技术力量的保障，一些危急患者常来不及进行临床救治就死亡了。通过超声造影引导的微创止血方法有止血剂局部注射法、经皮射频凝固法、微波凝固法、高强度聚焦超声法（HIFU）等。这些微创的方法可以获得较好的止血效果，并且恢复时间较短。但是他们都具有一定的局限性。其中，止血剂对级别较大的创伤出血疗效并不满意，且会引起疼痛。其他三种方法都是通过局部升温致组织产生凝固样坏死来实现止血目的。但是当局部靶组织温度迅速升达70℃以上时，易引起实质脏器或周围组织结构变形。而且，因为存在热沉效应使治疗效果不佳或治疗时间延长。另外，上述止血法只能实现点对点的止血，这对较大范围的出血面治疗具有一定的局限性。而HIFU治疗过程中，空化效应可以促使凝血块或均质物形成，使温度升高至80℃以上，从而加强热效应止血作用。超声联合造影剂微泡不仅能引导上述微创止血过程，而且激发的空化效应可毁损微小血管而阻断内脏器官的血流（即血管毁损术），从而实现对肝、脾整齐切割伤的止血作用。然而，这些切割伤为I级损伤，其创面整齐，出血量较少且不稳定，无法排除两创面相接触而形成的压迫性止血及局部血凝块形成止血作用。以上述研究为基础，本实验对肝组织的另一创伤类型——肝脏断裂伤（Ⅱ级外伤）进行止血治疗，通过改善非聚焦性脉冲超声空化治疗仪参数，进一步探讨超声联合微泡治疗的可行性及病理机制。目的超声联合微泡可以阻断正常肝脏、脾脏的血液循环，对肝、脾整齐的切割伤辐照治疗后，其创面出血明显减少。但上述创伤止血实验的模型为Ⅰ级外伤，无法排除两创面相接触形成的压迫性止血及局部血凝块形成的止血作用。基于以上研究，本实验建立二级外伤模型——兔肝断裂伤，通过观察新型脉冲式超声空化治疗仪联合微泡造影剂脂氟显对其创面的止血疗效，进一步探讨超声联合微泡作为一种无创方法治疗肝断裂伤的可行性以及止血的病理学机制。方法健康成年雄性新西兰大白兔14只。14只实验新西兰大白兔随机分成3组，即超声微泡组(MEUS)6只、单纯治疗超声组(TUS)4只、单纯微泡组(MB)组4只。建立肝断裂伤模型：麻醉实验兔并建立静脉通道，将兔固定于实验台，开腹后并将肝脏牵拉出腹腔，剪断肝脏左叶远端制作肝断裂伤模型。分组实验方法：（1）MEUS组：用治疗探头直接垂直辐照伤口创面5min，治疗开始的4min内静脉匀速推注造影剂微泡，治疗时缓慢匀速移动探头以均匀辐照全整个伤口；（2）TUS组：经耳缘静脉匀速推注2ml生理盐水替代造影剂微泡，同时使用脉冲式超声治疗仪以同样的方法辐照肝创面；（3）MB组，经耳缘静脉通道4分钟内推注相同剂量的造影剂微泡，同时超声治疗仪探头使用同样的方法置于肝创面假照（但不发射脉冲）。创伤出血评分方法：（1）视觉评分法：辐照前后，通过肉眼观察，对创面出血情况进行观察并评分。（2）称重法：辐照前后，用吸水滤纸收集创面30s出血量，精密天平进行称重，各组辐照前后出血的差值为创面出血的净重。超声造影灌注情况：辐照治疗前后均对实验肝进行超声造影，观察靶区的造影剂灌注情况进行分级，并进行声学密度分析（PI,AUC）。病理学检查：治疗结束后，随机抽取MEUS组三只实验兔及所有对照组进行病理学检查，余下MEUS组三只实验兔抗感染饲养48小时后取辐照靶区的组织进行病理学检查。统计学方法：辐照前后出血速度、声学密度分析（PI、AUC）的数据以平均数Mean±SD表示，辐照前后的出血视觉评分以中位数Median（Q25，Q75）表示。各组辐照前后的出血速度、峰值强度（PI）、曲线下面积（AUC）的组内比较采用配对t检验（Paired-Samples T Test），各组之间对应的辐照前后出血速度、PI、AUC的组间比较采用单向方差分析(one-way ANOVA)及多个均数间两两比较的LSD法进行检验。各组出血视觉评价辐照前后的组内比较采用Wilcoxon秩和检验，各组之间对应的辐照前后出血视觉评分组间比较采用Kruskal-Wallis H test及Mann-Whitney U进行检验。辐照前后出血速度与PI、AUC之间，PI与AUC之间采用Pearson分析。辐照前后出血速度与出血视觉判断之间采用Spearman分析。P0.05表示为差异有统计学意义。所有数据均采用SPSS13.0软件分析。结果一、止血效果 1．出血视觉评价：MEUS组辐照治疗后即刻观察，出血视觉评分下降，由4（3,4）分降至1（0.75,1），与治疗前比较差异有统计学意义（P0.05）；分别与TUS组、MB组治疗后比较差异有统计学意义（P0.05） 2．出血速度评价：将肝创面30s的出血总量除以30s得到平均出血速度。辐照前所有实验肝出血速度计量基本一致，三组数据两两比较差异无统计学意义（P0.05）。辐照后，MEUS组出血速度明显下降,由0.013±0.004降至0.002±0.001g/s，与治疗前比较差异有统计学意义（P0.05）；分别与TUS组、MB组治疗后比较差异有统计学意义（P0.05）二、超声造影 1．视觉判断：治疗前各组肝脏对应区域、TUS组和MB组的辐照区域的造影剂灌注良好，评分为0级。MEUS组辐照后可见造影剂沿着肝大血管稍缓慢不均匀地进入肝组织中，呈明显大小不等不规则的充盈缺损区，，缺损区的边缘灌注较低，评分为Ⅱ级。 2．声学密度分析：辐照前各组峰值强度、曲线下面积比较差异无显著意义（P0.05）。辐照后MEUS组的峰值强度（PI）由22.000±6.090%降至9.517±1.380%，曲线下面积(AUC)由2173.983±839.190%s降至872.583±196.409%s，差异有统计学意义（P0.05）；分别与TUS组、MB组辐照后的组间比较差异有统计学意义（P0.05）。 3．相关性分析：肝创面辐照后出血速度分别与曲线下面积、峰值强度之间呈正相关(P0.05)；辐照前后的峰值强度与曲线下面积之间、辐照后视觉评分与出血速度之间呈正相关(P0.05)。三、病理所见光镜下，TUS组、MB组辐照后肝索、肝板结构清楚，肝窦大小均匀，肝窦间见散在红细胞分布。MEUS组辐照后病变区域四种病变混合存在，主要表现为：1肝细胞浑浊肿胀，压闭窦状间隙及肝窦间隙，以临近创伤面明显；2广泛的窦状间隙、肝窦间及小静脉内出现了大量的红细胞淤积，此为稍远离创伤面的主要表现；3部分汇管区结缔组织可见明显出血；4肝组织出现散在的小灶样坏死，并见少量炎细胞浸润。MEUS组饲养48小时后肉眼见肝脏创伤面灰白，光滑无出血（即出血速度视觉评分0分）；光镜下所见，在距创伤面约1.5cm范围内的坏死灶较治疗后即刻增大，坏死灶外周为变性细胞带包绕，变性细胞区见少许炎细胞浸润，病变区域呈地图样。病变区域与正常肝组织间存在明显的分界，靠近病变区域肝组织的肝窦内可见少量红细胞分布。结论 1、超声联合微泡可以有效地阻断肝脏局部血液循环。 2、超声联合微泡对肝断裂伤辐照治疗，使创面出血量减少10倍以上，超声联合微泡有潜力成为临床治疗肝脏断裂伤（Ⅱ级损伤）的一种新型、有效、无创的止血治疗方法。 3、超声联合微泡可以实现创面几乎停止，出血主要机制可能是肝细胞与小血管壁不同程度损伤，肝窦及窦间隙受压及汇管区周围组织出血压迫等致使靶区血管的血流停滞或阻断。 4、本研究使用的超声治疗仪及造影剂微泡轻巧便携，有潜力成为临床应用于现场急救的一种新方法。
[Abstract]:background
Liver tissue is fragile, blood flow is very rich, and its volume is large and its location is relatively fixed. Therefore, it is easy to break and break when it is attacked by violence.
First aid to cope with liver rupture, surgical treatment is mainly used in clinic, including gauze block filling, electrocoagulation hemostasis, argon gas hemostasis, simple suture repair, hepatic artery embolization or ligation and partial hepatectomy. Although the effect is significant, there are large surgical injuries to the patients, and surgical treatment often requires certain equipment and technique. These minimally invasive methods, such as local injection of hemostatic agents, percutaneous radiofrequency coagulation, microwave coagulation, high intensity focused ultrasound (HIFU), and so on. These minimally invasive methods can obtain better hemostatic effects and recover time. It is short, but they all have some limitations. Among them, the hemostatic agent is not satisfied with the higher level of traumatic bleeding and causes pain. The other three methods are caused by the local warming to produce coagulation necrosis to achieve the aim of hemostasis. But when the temperature of the target tissue of the authorities rapidly rises above 70 centigrade, it is easy to cause the substance. The structure of the viscera or surrounding tissue is deformed. Moreover, because of the existence of heat sink effect, the treatment effect is not good or the time of treatment is prolonged. In addition, the above hemostasis can only achieve point to point hemostasis, which has certain limitations for a large range of bleeding surface treatment. In the course of HIFU treatment, the cavitation effect can cause the formation of clots or homogenate, Raising the temperature to above 80 degrees, thereby enhancing the heat effect of hemostasis.
Ultrasound combined with contrast media can not only guide the above-mentioned minimally invasive hemostasis, but also stimulate the cavitation effect to destroy the tiny blood vessels and block the blood flow of the viscera (vascular damage), thus achieving the hemostasis effect on the liver and spleen neatly cut. However, these cuts are I injury, the wound is neat, the amount of bleeding is less and unstable. The oppressive hemostasis and local blood clot formed by two facial contact can not be excluded. Based on the above study, the experiment has done a hemostatic treatment for another type of liver injury, the liver fracture injury (grade II trauma), and further explores the ultrasonic combination by improving the parameters of the non focused pulse ultrasonic cavitation therapy instrument. The feasibility and pathological mechanism of combined microbubble therapy.
objective
Ultrasound combined with microbubbles can block the normal liver, the blood circulation of the spleen, and the wound bleeding of the liver and spleen neatly cut after irradiation treatment, the bleeding of the wound is obviously reduced. However, the model of the trauma hemostasis experiment is a grade I trauma, which can not exclude the hemostatic effect of the compression hemostasis and the formation of local blood clot formed by two facial contact. Based on the above research, In this experiment, we established a two stage model of traumatic injury of rabbit liver. By observing the hemostatic effect of the new pulse ultrasonic cavitation therapy instrument combined with microbubble contrast agent lipofluorine, the feasibility of ultrasonic combined microbubble as a noninvasive method for the treatment of liver fracture and the pathological mechanism of hemostasis were further explored.
Method
A healthy adult male New Zealand white rabbit 14.14 rabbits were divided into 3 groups randomly, that is, 6 ultrasound microbubbles (MEUS), 4 only treated group (TUS), and 4 group of simple microbubble group (MB).
The model of liver fracture injury was established: the rabbits were anaesthetized and the venous channel was established. The rabbit was fixed on the experimental platform. After opening the abdomen, the liver was pulled out of the abdominal cavity, and the model of liver broken laceration was made by cutting the left lobe of the liver.
Group experiment methods: (1) group MEUS: directly irradiate the wound surface 5min with a therapeutic probe, treatment of the initial 4min intravenous injection of contrast agent microbubbles at the beginning of the treatment, and the slow and uniform moving probe at the time of treatment to irradiate the whole wound evenly; (2) group TUS: 2ml physiological saline was injected into the auricular vein to replace the contrast agent microbubbles, and the pulse was used at the same time. The same method was used to irradiate the liver wound with the same method. (3) in group MB, the same dose of contrast microbubbles were injected within 4 minutes of the auricular vein channel, and the ultrasonic therapeutic probe was used in the same method for the liver wound (but not the pulse).
The scoring method of traumatic bleeding score: (1) visual scoring method: before and after irradiation, the bleeding of the wound was observed and graded by the naked eye. (2) before and after irradiation, the amount of 30s bleeding was collected with the absorbent filter paper, the precision balance was weighed, and the difference between the blood and the blood was the net weight of the wound bleeding before and after irradiation.
Ultrasound contrast perfusion: the experimental liver was contrast-enhanced before and after irradiation, and the contrast agent perfusion in the target area was observed and the acoustic density analysis (PI, AUC) was performed.
Pathological examination: after the treatment, three rabbits were randomly selected from the MEUS group and all the control groups were examined for pathology. The remaining three rabbits in the remaining group of the MEUS rabbits were reared for 48 hours, and the tissues of the irradiated target area were examined for pathological examination.
Statistical methods: the velocity of bleeding before and after irradiation, the data of PI (AUC) was expressed as an average of Mean + SD. The visual score of bleeding before and after irradiation was expressed as Median (Q25, Q75). The rate of bleeding, peak intensity (PI) before and after irradiation, and the area under the curve (AUC) were compared to the paired t test (Paired-Samples T). In each group, the velocity of bleeding before and after irradiation, the comparison of PI, AUC between groups using one-way ANOVA (one-way ANOVA) and the LSD method of 22 comparison among multiple mean numbers, each group of hemorrhagic vision was compared with Wilcoxon rank and test before and after irradiation, and the corresponding inter group ratio of visual scoring before and after irradiation was compared. Kruskal-Wallis H test and Mann-Whitney U were compared. The bleeding speed before and after irradiation, between PI, AUC, PI and AUC were analyzed by Pearson. The difference between the bleeding speed and the visual judgement before and after irradiation was statistically significant. All the data were analyzed by the software.
Result
First, the effect of hemostasis
1. visual evaluation of hemorrhage: immediate observation after irradiation treatment in group MEUS, the visual score of bleeding decreased from 4 (3,4) to 1 (0.75,1), and the difference was statistically significant (P0.05) compared with that before treatment (P0.05), and there was a significant difference between group TUS and group MB (P0.05).
2. evaluation of bleeding speed: the average bleeding rate was obtained by dividing the total amount of hemorrhage of 30s in the liver wound by 30s. All the experimental liver bleeding rates before irradiation were basically the same. There was no statistical difference between the three groups of data 22 (P0.05). After irradiation, the bleeding rate of the MEUS group decreased significantly from 0.013 + 0.004 to 0.002 + 0.001g/s, compared with the pre treatment comparison. The difference was statistically significant (P0.05), and there was significant difference between group TUS and group MB after treatment (P0.05).
Two, contrast-enhanced ultrasound
1. visual judgment: the contrast medium of the liver in each group before treatment, the contrast agent in the irradiated area of group TUS and group MB was well perfused, and the grade 0.MEUS group was irradiated, and the contrast agent entered the liver tissue slightly and slowly along the large blood vessels of the liver. Class II.
2. acoustic density analysis: there was no significant difference between the peak intensity of each group before irradiation and the area under the curve (P0.05). The peak intensity (PI) of group MEUS decreased from 22 + 6.090% to 9.517 + 1.380%, and the area under the curve (AUC) decreased from 2173.983 + 839.190%s to 872.583 + 196.409%s, and the difference was statistically significant (P0.05), respectively, with TUS group and MB group, respectively. The difference between groups after irradiation was statistically significant (P0.05).
3. correlation analysis: there was a positive correlation between the bleeding speed of the liver wound after irradiation and the area under the curve and the peak intensity (P0.05), and between the peak intensity before and after irradiation and the area under the curve, there was a positive correlation between the visual score and the speed of bleeding after irradiation (P0.05).
Three, pathological findings
Under the light microscope, group TUS and group MB irradiated the hepatic cord, the structure of the liver was clear, the size of the hepatic sinusoid was uniform, and the hepatic sinus was scattered in the red blood cell group.MEUS after irradiation, and four kinds of lesions were mixed in the lesion area. The main manifestations were: 1 the turbid swelling of the liver cells, the closed sinus space and the hepatic sinus space, which were adjacent to the traumatic surface; 2 extensive sinus spaces, and the hepatic sinusoids. A large amount of red blood cell deposition was found in the small vein, which was the main manifestation of a little far away from the wound surface; the 3 parts of the connective tissue of the 3 part of the pipe showed obvious bleeding, 4 liver tissue appeared scattered small focal necrosis, and a small amount of inflammatory cells infiltrated the.MEUS group after 48 hours to see the gray white of the wound surface of the liver. In the light microscope, the necrotic foci in the area of about 1.5cm of 1.5cm were increased immediately after the treatment. The necrotic foci were wrapped around the denatured cells, and the degeneration cell area was infiltrated with small inflammatory cells. The lesion area was map like. The lesion area and normal liver tissue were in distinct boundary, close to the hepatic sinusoid of the liver tissue in the lesion area. A small amount of red blood cell distribution was found.
conclusion
1, ultrasound combined with microbubbles can effectively block the local blood circulation in the liver.
2, ultrasound combined with microbubbles can reduce the amount of bleeding of the wound by 10 times. Ultrasound combined with microbubbles has the potential to be a new, effective and noninvasive method of hemostasis for the treatment of liver fracture injury (grade II injury).
3, ultrasonic combined with microbubbles can almost stop the wound. The main mechanism of bleeding may be that the liver cells and the small vessel wall are damaged in different degrees. The pressure of the hepatic sinusoid and the sinus space and the compression of the tissue around the tube area lead to the stagnation or blocking of the blood flow in the target vessels.
4, the ultrasound therapeutic apparatus and contrast agent microbubbles used in this study are portable and portable, and have potential to be a new method for clinical first-aid in the field.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.1;R657.3

【参考文献】