摘要
背景
肿瘤的治疗是医学界的难点和研究热点,对于无法手术治疗的肿瘤,局部高温热疗是主要的可选手段之一。由于肿瘤细胞对热更敏感,在相同温度下,肿瘤细胞对热效应更加敏感,而正常组织可不受损伤或受损伤较小,使热疗成为治疗肿瘤的重要方法。超声热疗是目前肿瘤热疗的主要方法之一,增强超声热疗效果的方法一般有:1.提高超声辐照能量,通过增加发射功率和延长辐照时间来实现,但过高的能量也可能对透声通路和靶区周围组织造成损伤;2.改变靶区组织声学特性,增加声阻抗,使能量在靶区更有效沉积。3.减少组织血流灌注,增加热能在局部的沉积。目前尚缺乏一种非创伤性阻断肿瘤局部血供的方法。
在热疗中机体热场的分布是治疗成功的关键,治疗过程中温度的控制是热场分布的重要决定因素。临床常用的微波、射频、激光等热疗方法,针距及每根针的有效范围不易精确控制,不能保证整块肿瘤均匀加热。这主要是因为血液循环造成的热散失使热效应温升无法有效控制,因此热疗的范围、升温过程及最高温度的控制等方面的问题还有待研究。
本研究前期试验发现微泡增强的超声空化效应可以造成正常兔肝脏小血管管壁空化机械损伤,出血、血肿和血栓形成,暂时性阻断局部血流,可能是一种新型非创伤性阻断肝脏局部血供的方法。
目的
本研究建立在超声增强的微泡空化阻断兔正常肝脏血流基础上,研究目的是探讨暂时性阻断肝脏血流灌注对肝脏超声热疗的促进作用,并分析可能造成的肝损伤副作用。
材料与方法
一、主要仪器及试剂
1.实验仪器:第三军医大学新桥医院研制的脉冲式聚焦超声空化治疗仪治疗头频率831KHz,发射占空比0.5%,峰值负压为4.6 MPa,平均声强(ISPTA)0.89 W/cm2。GE公司Logic 9彩色多普勒超声仪,配有超声造影模式。澳大利亚Metron(美康公司)生产的超声波治疗仪。广州市圣高测控科技有限公司生产的测温仪。
2.实验试剂:第三军医大学新桥医院研制的“脂氟显”脂质微泡,乳白色凝乳状微泡悬浮液,核心气体为全氟丙烷,微泡浓度约为(4-9)×109/ml,98%粒径小于8μm,平均2μm。
二、实验方法
40只健康新西兰大白兔,随机分为4组,分别为超声联合微泡加热疗组(USC+MB+UST)、单纯微泡加热疗组(MB+UST)、生理盐水加热疗组(NS+UST)、单纯微泡+假照组(MB+Sham),每组各10只。开腹暴露肝脏部分左中叶后,各组分别处理如下:
(一)超声空化
1.USC+MB+UST组:经耳缘静脉注射脂质微泡造影剂,同时实施脉冲式超声空化辐照其肝脏5 min。
2.MB+UST组:经耳缘静脉推注脂质微泡造影剂,但不实施脉冲式超声空化。
3.NS+UST组:经耳缘静脉推注生理盐水,但不实施脉冲式超声空化。
4.MB+Sham组:处理与MB+UST组相同。
(二)热疗过程
各组经以上方案处理后,在超声热疗处理前,在被阻断的肝脏局部插入测温电极,测温仪每隔十秒记录一次温度变化。1、2、3组立即用超声热疗仪持续热疗照射被阻断的局部肝脏,在温度达到42℃后,再持续热疗10 min,测温时间约20 min;4组实施超声热疗假照(不发射超声热疗能量),测温时间与前几组相当,约20 min。
(三)超声造影、肝功检查和病理检查
第1、2、4组在超声空化阻断血流前、阻断后以及超声热疗后三个时间点实施超声造影观察局部肝脏血流变化。第3组在仅热疗后实施对肝脏超声造影。各组在超声空化阻断前、阻断后和超声热疗后三个时间点抽取静脉血检查肝酶谱、出凝血指标变化。术后取处理部分的肝脏组织作HE染色,光镜观察肝细胞、血窦的改变。
(四)分析方法
1.视频密度分析法:记录各组在治疗前后各时间点,靶区超声造影动态影像。以JPG格式输出各感兴趣点的超声造影高峰数字化图像;然后采用Adobe Photoshop CS3软件中的直方图功能对其肝脏治疗感兴趣区域(ROI)进行灰阶定量分析,取样注意避开大中血管断面,软件自动计算出取样框内的平均灰阶值(GSV)。
2.实时记录温度变化,计算最终温度变化幅度,描绘温度变化曲线,病理学检查肝脏血管、细胞改变。
结果
一、视频密度
治疗前每组各时间点的超声造影高峰平均灰度值,肝脏ROI区域的平均灰度值(GSV)范围是116.42±6.18~119.52±4.14℃,三组差异无统计学意义。治疗后,USC+MB+UST组局部肝脏血流灌注被空化阻断,在治疗后0min,其ROI区域造影高峰平均灰度值显著降为0.42±0.29℃,热疗后为2.18±0.68℃,空化治疗前与治疗后比较差异具有统计学意义;而MB+UST组、NS+UST组和MB+Sham组所有治疗前后的时间点,实施超声造影高峰的平均灰度值变化在112.98±7.73~118.30±3.87℃内,组内治疗前后造影高峰GSV比较差异无统计学意义,但是,与USC+MB+UST组治疗后的GSV比较差异具有统计学意义。
二、温度变化
USC+MB+UST组热疗后温度平稳上升,最终温度上升幅度最大(均数,以下同)达17.81±2.41℃,即热疗效果最明显,达到42℃所需时间7.43±1.49min(分钟)、与MB+UST组所需时间7.00±5.17min无明显差异(P>0.05),温度变化曲线与方程拟合度最高,回归系数:线性方程R=0.909二次曲线方程R=0.924 ;MB+UST组最终温度上升幅度达13.23±5.89℃,与USC+MB+UST组比较差异无统计学意义(P>0.05),温度变化曲线与方程拟合度较差,回归系数:线性方程R=0.259,二次曲线方程R=0.277。NS+UST组最终温度上升幅度为7.11±2.78℃,多组未能达42℃,温度变化曲线与方程拟合度比USC+MB+UST组差,回归系数:线性方程R=0.632,二次曲线方程R=0.639;MB+Sham组最终温度上升幅度为-0.68±0.88℃,不能达42℃,温度变化曲线与方程拟合度最差,回归系数:线性方程R=0.115,二次曲线方程R=0.119。
各组均同时记录自身未处理部分肝脏的温度变化,1、2、3组热疗后比未热疗前略有上升,幅度在3.15±1.36~3.88±2.13℃范围,其温升幅度显著低于自身肝脏超声处理部分温升(P<0.01),但三组组间比较无明显差异;MB+Sham组的自身温度变化为-0.94℃,两者无明显差别(P>0.05)。
三、肝功检查结果
各组肝酶谱均有上升,USC+MB+UST组肝酶谱升高程度显著高于其它各组,差异有统计学意义(P<0.05);出凝血指标无明显差异(P>0.05)。
四、病理改变
USC+MB+UST组:热疗组织汇管区充血、出血、血栓形成,动静脉管壁被损伤,肝窦大量充血,肝细胞浑浊肿胀,部分组织内可见空泡分布。MB+UST组:热疗组织汇管区充血,局部少量出血,动静脉管壁相对完整,肝窦轻度充血,肝细胞浑浊肿胀,部分组织内可见空泡分布。NS+UST组:部分管腔轻-中度充血,无出血,部分肝细胞轻度浑浊肿胀,未见空泡。MB+Sham组:汇管区管腔无充血、出血,肝窦见少量红细胞散在分布,肝细胞无明显改变。
结论
在微泡增强的超声空化阻断兔正常肝脏血流的基础上,实施超声热疗可以获得比非循环阻断情况下更显著的温升效果;单纯微泡的存在也具有一定促进超声热疗温升的作用;微泡增强的超声空化治疗在阻断肝脏血液循环时,产生了一定程度的肝损伤,表现为肝酶谱显著上升,但出凝血指标无明显变化。
Background
Cancer treatment is known as a difficult and hot research in the medical field. For the treatment of inoperable tumors, local high temperature thermotherapy is one of the primary means of treating the liver cancer. As the tumor cells are more sensitive to heat, at the same temperature, normal tissue can be free of or less of injury or damage, which makes thermotherapy an important method of cancer treatment. Now, the common ways of enhancing the effects of thermotherapy are as follows: First, improve the radiation energy, which is achieved by increasing the transmission power and extending irradiation time. But the over high energy may also cause damage towards the sound transmission path and the surrounding tissues. Second, change the acoustic properties of the target tissues and enhance acoustic impedance to improve the effect of energy deposition in the target area. Third, reduction of tissue blood perfusion, increase the heat energy in the local area. But by now, a way of blocking the blood supply towards the tumor area has not been found yet.
During the thermotherapy, the thermal distribution is the key to the successful treatment, and the control of temperature is an important determinant of the thermal distribution. For the commonly used laser for microwave frequency generation in clinic, the needle distance and the effective range of each needle is not easy to control precisely, so that it can not guarantee that the tumor be heated in a balanced way. Because of heat loss by blood circulation,heating effect out contral .Therefore, the scope of thermotherapy, the process of heating, the control of the maximum temperature and other issues are still to be studied.
Previous study found that cavitation ultrasonic caused by micro-bubble can separate the mechanical damage from the small blood vessels wall in normal rabbit liver , to cause bleeding, hematoma and thrombosis and therefore to block the blood flow temporary. During this experiment, maybe a new non-invasive embolism was used to block the local blood supply. This method can be free of the influence of the blood circulation, which can remove heat to cause the temperature increase. And then, through the Thermotherapy, study whether blocking blood flow can strengthen the effect of ultrasonic Thermotherapy.
Objective
This study is based on micro-bubble caused ultrasound cavitation blocking normal rabbit liver blood flow. The purpose of the study is to discuss the effect of the temporary blocking of liver blood perfusion on the liver ultrasound thermotherapy, and to analyze the possible damage towards liver.
Materials and Methods
1.Principal instruments and reagents
1. Experimental instruments:
The small, pulsed, focused ultrasonic cavitation treatment apparatus developed by Xinqiao Hospital of the Third Military Medical University, with a treatment probe frequency of 831KHz, an emission duty of 0.5%, a peak negative pressure of 4.6 MPa, a mean sound intensity of (ISPTA)0.89 W/cm2. Logic 9 color Doppler ultrasonic machine with ultrasonic contrasting mode (GE). Ultrasonic therapeutic apparatus of Australia Metron (Meikang Corporation) Thermodetector of Guangzhou Shenggao Observe and Control Technology Co. Ltd.
2.Experimental reagents:
“Zhifuxian”, a kind of lipid microbubbles in milky suspension developed by Xinqiao Hospital of the Third Military Medical University, with perfluoropropane as core gas at approximately(4-9)×109 microbubbles/ml. 98% microbubbles <8μm (mean, 2μm) in diameter.
3.Methods
40 healthy New Zealand rabbits were randomly divided into 4 groups, each for ultrasound and microbubble with thermotherapy (USC + MB + UST), a simple micro-bubble with thermotherapy (MB + UST), saline with thermotherapy (NS + UST),and a simple micro-bubble + sham thermotherapy control group (MB + Sham). 10 in each group. Open the abdomen to explore the left middle liver, then each group was treated ultrasonic Cavitation and thermotherapy as follows:
(1) Ultrasonic Cavitation
1) USC + MB + UST Group: inject lipid microbubbles contrast medium via ear vein with 5 minutes of pulse ultrasonic irradiation towards the liver.
2) MB + UST Group: inject lipid microbubbles contrast medium via ear vein with no pulse ultrasonic irradiation
3) NS + UST group: inject physiological saline via ear vein with no pulse ultrasonic irradiation
4) MB + Sham group: inject lipid microbubbles contrast medium via ear vein with no pulse ultrasonic irradiation
(2) Thermotherapy
After the treatment of Ultrasonic Cavitation, each group uses part of the liver where blood flow has been blocked through thermotherapy of ultrasound therapy apparatus. Them insert the temperature electrode,Every 10 seconds, write down the temperature by using thermodetector. After the temperature reaches 42℃, it continued to hyperthermia for 10 minutes, all for about 20 minutes; MB + Sham groups according to the implementation of ultrasound hyperthermia leave (not fired ultrasound hyperthermia energy), temperature measurement time about 20 minutes also .
(3) Contrast Enhanced Ultrasound Liver function tests and Pathology
As for the Group 1, 2 and 4, observe the change of the liver blood flow before the block, after the block and after the thermotherapy by Contrast Enhanced Ultrasound. But group 3 having CEUS only after the thermotherapy.
Take the venous blood of each group before the block, after the block and after the thermotherapy to observe the liver enzymes. After the operation, take the treated liver tissue as HE staining, observe the liver cells and sinusoids under the light microscopy.
(4) Analyses
1) Visual density analysis: The dynamic ultrasonic contrasting images of the target area were recorded at various time points in each group. The digital images of points of interest were imported in the JPG format, and then were subjected to quantitative grayscale analysis using the histogram feature of Adobe Photoshop CS3 software. The cross-section of large or medium vessels should be avoided during sampling. The mean grayscale value in the sampling window was calculated automatically by the software.
2) Record the temperature change, calculate the final temperature range, depict temperature variation curve, and pathologically examine the change of liver blood vessels and cells.
Results
1.Visual density
Before treatment, in USC + MB + UST group, the mean grayscale value and ROI contrast mean grayscale value 116.42±6.18~119.52±4.14differed insignificantly between the 1.2.4groups at various time points. in the three groups; 0min after the cavitation treatment, in the USC + MB + UST group, the peak gray value of ROI was significantly reduced by 0.42±0.29 ~ 2.18±0.68 after thermotherapy, while for MB + UST group, NS + UST group and the MB + Sham group, whenever the time is, the average gray value is within 112.98±7.73~118.30±3.87, there was statistical difference in the groupitself.
2.Temperature change
In USC + MB + UST group, the temperature rose steadily after thermotherapy, eventually the maximum temperature rise (mean, same below) up by 17.81±2.41℃, which means the most obvious effect of Thermotherapy. It took 7.43±1.49min (minutes) to reach 42℃, there was no significant difference with the MB + UST group, which took 7.00±5.17min (P>0.05), the temperature curve was most fitted with the equation, regression coefficient, linear equation R = 0.909 the quadratic curve equation R = 0.924; MB + UST group’s final temperature rise up by 13.23±5.89℃, compared with the USC + MB + UST group P = 0.159, temperature curve was not well fitted with equation, regression coefficient, linear equation R = 0.259 the quadratic curve equation R = 0.277; NS + UST group’s final temperature rose up by 7.11±2.78℃, and many groups failed to reach 42℃, the temperature change curve was poor to fit with the equation, regression coefficient, linear equation R = 0.632 the quadratic curve equation R = 0.639; MB + Sham group‘s final temperature rise by-0.68±0.88℃, and can not be up to 42℃, temperature curve was the worst to fit with the equation, regression coefficient, linear equation R = 0.115 the quadratic curve equation R = 0.119. At the same time, each groups recorded the temperature of the non treated liver, in the first three groups, the temperature increased slightly than the one before Thermotherapy (3.15±1.36~3.88±2.13), the range of change was far less than treated parts (P <0.01), there was no significant difference among the three groups; MB + Sham group’s temperature reduced by 0.94℃on their own, there was no significant difference (P>0.05).
3.Liver function tests
Liver enzymes increased, and there was difference among the groups (P <0.05 , mainly caused by the time factor, the experimental treatment group was also a factor; no significant differences had been found in the coagulation (P>0.05).
4.Pathological changes
USC + MB + UST Group: Thermotherapy portal area had tissue hyperemia, hemorrhage and thrombosis, and the wall of Sucquet's canal was injured, there was massive haemorrhage in sinus hepaticus. The liver cells were opaque and tumorous, there were some vacuoles distributed in some tissues. MB + UST group: Thermotherapy portal area had tissue hyperemia, less hemorrhage in some parts; the wall of Sucquet's canal was relatively complete; there was less hyperemia in sinus hepaticus, and the liver cells were opaque and tumorous, there were some vacuoles distributed in some tissues. NS + UST Group: there were light - moderate hyperemia in some lumens, no bleeding, and some liver cells were less opaque and tumorous. There was no vacuole found. MB + Sham group: there was no hyperemia or hemorrhage in the lumens of the portal area. Sinus hepaticus saw a small amount of scattered red blood cells. There was no significant change in the liver cells.
Conclusions
The cavitation effect resulting from the use of microbubbles and a pulsed ultrasonic cavitation treatment apparatus can block normal liver microcirculation temporarily, Thermotherapy had achieved more significant temperature rise under the foundation than no-cycling blocked . Simply the existence of micro-bubble has a certain temperature to promote the role of ultrasound hyperthermiaTemperature also. After the whole block of the blood flow, the side effect of the treatment was the increase of liver enzymes. There was no significant change in the coagulation index.
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