WO2013053099A1 - 微泡联合超声空化的肝创伤止血用途 - Google Patents

微泡联合超声空化的肝创伤止血用途 Download PDF

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WO2013053099A1
WO2013053099A1 PCT/CN2011/080620 CN2011080620W WO2013053099A1 WO 2013053099 A1 WO2013053099 A1 WO 2013053099A1 CN 2011080620 W CN2011080620 W CN 2011080620W WO 2013053099 A1 WO2013053099 A1 WO 2013053099A1
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ultrasound
cavitation
microbubble
hemorrhagic
liver
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PCT/CN2011/080620
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French (fr)
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刘政
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Liu Zheng
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N7/02—Localised ultrasound hyperthermia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0039—Ultrasound therapy using microbubbles

Definitions

  • the invention relates to a new use of hemostatic treatment under the excitation of ultrasound, which can be applied to the post-traumatic hemostasis treatment of liver parenchymal organs, and belongs to the technical field of non-invasive ultrasound treatment.
  • liver is the most vulnerable organ in abdominal trauma, and post-traumatic bleeding is the most common complication after abdominal trauma, and hemorrhagic shock is the leading cause of death.
  • Liver blood supply is rich and soft body organs, about 50% of liver rupture and chronic oozing still need surgery to stop bleeding [1 2] , complex liver trauma is not easy to repair and suture, and the surgery is traumatic [ 3] .
  • ultrasound contrast-guided hemostatic injection, microwave or radiofrequency thermocoagulation hemostasis, high-intensity focused ultrasound to stop bleeding, interventional vascular embolization, etc. have been developed, the above hemostasis methods may have limitations in hemostasis and complicated equipment. The need for precise image navigation, long time and other limitations, these treatments often can not meet the patient's treatment requirements, so it is necessary to find a non-traumatic method of hemostasis after abdominal trauma.
  • High-intensity focused ultrasound uses the thermal effect of ultrasound to perform hemostasis in liver trauma.
  • HIFU liver hemostasis needs to be performed under open eyes, while deep bleeding requires accurate image guidance to focus ultrasound energy.
  • This method requires a large ultrasound device, an anesthesia is required during the operation, and the operation is complicated.
  • acoustic impedance interface blocking such as ribs or intestinal tract, poor aggregation of thermal effects, uneven conduction, etc., which may burn vital organs such as skin, ribs and intestines, causing serious side effects.
  • the treatment trial has never been implemented. Its hemostasis is mainly limited to the surface of the liver, and the treatment time is long. It is greatly restricted in the treatment and has not been applied in clinical practice [4 _ 7] .
  • the cavitation effect refers to a series of kinetic processes in the liquid that cause oscillation, expansion, contraction and implosion under the action of ultrasound, accompanied by transient high temperature and high pressure. , multiple shock release behaviors such as shock waves, discharges, and microjets. [8] is another thermal effect. A major physical effect of ultrasound.
  • Hwang et al. used intravenous injection: foam combined with focused ultrasound cavitation damage to achieve damage to rabbit rim vein vascular endothelium, but requires local intraluminal Injection of small doses of thrombin may cause vascular embolization;
  • Cain " in the cavitation study does not use intravenous microbubbles, but uses ultrasound to stimulate the body's own potential microbubbles to undergo cavitation for tissue ablation. Tissues such as normal normal myocardium are the research objects; Mi ller et al.
  • the invention provides a novel non-invasive use of ultrasonic cavitation hemostasis in the preparation of combined ultrasound to produce cavitation as a hemorrhagic hemorrhagic drug for hepatic trauma.
  • the microbubble is a microbubble capable of generating high-intensity acoustic cavitation under excitation of an ultrasonic pulse of a certain energy in a cycle; the cavitation can mechanically destroy tiny blood vessels of the local liver, forming perivascular hemorrhage, hematoma and Intravascular thrombus; the sonic effect of cavitation also leads to hepatocytes Swelling, squeezing the hepatic sinus, resulting in temporary circulatory blockage of the local liver tissue, resulting in hemostasis during liver trauma.
  • the microbubbles are selected from the group consisting of ultrasound contrast microbubbles or therapeutic microbubbles, selected from SonoVue, Def inty, O tisonTM, SonazoidTM, Imagent, perfluoropropanoid human albumin microspheres, and 0 ⁇
  • the above-mentioned hemorrhagic hemorrhage after liver trauma refers to the hemorrhage caused by the liver under various trauma conditions, including war wounds and common hemorrhage after various traumas of the abdomen.
  • the above ultrasonic energy is a pulsed or intermittent pulsed ultrasonic emission having a high peak sound pressure and a low emission duty ratio.
  • the above ultrasonic energy form its emission frequency ranges from 300 kHz to 3.0 MHz; the peak peak sound pressure including peak positive pressure and peak negative pressure is 1-9 MPa; each ultrasonic pulse width is 20 cycles Above; the total time of each pulsed ultrasonic emission treatment is more than 30 seconds; the pulsed ultrasonic generation can be continuous or intermittent, and the intermittent or pause time is 0-20 seconds; 0. 01-10% between.
  • the above-mentioned high-intensity acoustic cavitation refers to a strong acoustic cavitation effect which occurs when the microbubbles are excited by the ultrasonic energy form irradiation described in claim 5.
  • the therapeutic device that achieves the above-described ultrasound emission is a variety of forms of planar emission or focused emission ultrasound systems.
  • The::bubble-enhanced ultrasonic cavitation hemostasis treatment the hemostatic effect can be further enhanced when the prothrombin complex or fibrinogen is used in combination with the vein.
  • Ultrasound contrast enhancement of microvesicles has been approved in clinical diagnostics and is a new therapeutic use in the present invention.
  • the microbubbles which enter the blood circulation through the vein may have a high-intensity acoustic cavitation effect, which may first cause significant mechanical damage, hemorrhage and hematoma formation of the hepatic microvascular, thereby generating
  • the temporary (blood perfusion) blocking effect of the blood circulation blocks the source of bleeding and stops bleeding. It is characterized by blocking blood flow irrigation Note whether it is surface or deep tissue.
  • the ultrasonic energy of the present invention refers to a form of ultrasonic energy of a pulsed or intermittent pulse type, a higher peak sound pressure, and a lower emission duty ratio, and the frequency of the emission is in the range of 300 KHZ-3. OMHz; Continuous pulse or intermittent pulse type, intermittent (pause) time varies from 2-20 seconds; ultrasonic peak negative pressure is between 2-10 MPa; ultrasonic emission each pulse width is more than 20 cycles; ultrasonic emission duty cycle The change is between 0. 01-10%; thus the average sound intensity (I SPTA ) is less than 2 W/cm 2 , and the thermal effect is small. Each treatment time is between 30-600 seconds.
  • the acoustic parameters used in this study are ultrasonic emission frequency 831KHz, peak sound pressure 4. 47MPa, pulse width 400 cycles, pulse repetition frequency 9Hz, intermittent pulsed ultrasonic emission (emission 6 seconds / interval 6 seconds), actual duty
  • the average sound intensity is only (I SPTA ) 0. 4 W/cm 2 .
  • Figure 1 is a schematic diagram of the blood process
  • A The ruler scale is used as a standard, and a 2cm incision is cut with a scalpel.
  • B Collect 10 seconds of bleeding and calculate the initial bleeding rate.
  • C The therapeutic instrument probe covers the wound wound and performs hemostasis treatment.
  • D Hemorrhage hemostasis after treatment in the ultrasound treatment group.
  • Figure 2 is a comparison of visual assessment (A) and total bleeding volume (B) between the ultrasound microbubble group, the simple ultrasound group, and the sham group;
  • FIG. 1 Ultrasound observation of ultrasound ⁇ : Follicle group and sham group liver (AD), the anterior image before treatment showed that the two groups had good liver parenchymal filling (BE), and there was no difference before and after angiography in the sham group (BC). Congestion defect (EF) of contrast-enhanced ultrasound after hemostatic treatment in the ultrasound group;
  • FIG. 1 Schematic diagram of liver tissue structure after hemostasis.
  • the acoustic parameters of the ultrasonic therapy instrument include ultrasonic frequency, peak negative pressure, pulse width, average sound intensity (ITM) and duty ratio, etc., which are always measured by hydrophone method.
  • the received signal is received by a needle-shaped hydrophone (NTR1000, USA) 3 cm from the transmitting transducer, and amplified by a preamplifier (NTR, USA) by a digital oscilloscope (Ag i lent 55310, United States) Computer sample analysis. Measurement result,
  • OBJECTIVE To establish a model of rabbit liver injury and hemorrhage using ultrasound combined with microbubble cavitation effect.
  • New Zealand white rabbits 20 healthy New Zealand white rabbits, male or female, weighing 2000-2225g. New Zealand rabbits were anesthetized with Suspension New II 0. 2mg/kg, and fixed on the self-made planks on the supine side to establish the ear vein channel. 2% pentobarbital sodium (0.2 ml / kg) was anesthetized by ear vein injection.
  • the xiphoid process is prepared from the lower abdomen of the rib arch. The skin is disinfected according to the surgical aseptic procedure. The disinfection is performed on the skin. The laparotomy is performed under aseptic conditions. The 1 cm below the xiphoid is 1 to 2 cm along the abdominal white line.
  • the median incision was made by layer-by-layer laparotomy. After the liver was surgically exposed, the left middle lobe and the right liver were gently pulled out of the abdominal cavity, and fixed with saline infiltrated gauze. A 2 X 0. 5 cm wound was cut with a surgical blade (Fig. 2A), resulting in a hepatic wound injury model (Fig. 2B).
  • simple ultrasound group only ultrasound irradiation was performed; in the sham group, only the ultrasound treatment head was given.
  • the initial bleeding rate (ml / min) is the ratio of the amount of bleeding after the gauze suction incision is divided by the 10 s time. Take the original weight of the sterile gauze, the weight is accurate to 0. Olg, the timepiece is immediately followed by the stopwatch, while the gauze is sucking blood, after 10s, the gauze is weighed and the original weight of the gauze is obtained. 10s bleeding volume.
  • Ultrasound contrast was performed with coded excitation harmonic imaging, mechanical index 0. 13 , contrast group injection "lipid fluoride” microbubbles 0. 01 ml / kg, image depth, gain remained unchanged in the experiment.
  • the liver of the injured area was irradiated, and the ultrasound group: the blister group was treated with ultrasound irradiation combined with the vein: bubble injection; the ultrasound group only had ultrasound irradiation; the sham group only applied the ultrasound treatment head sham .
  • the microbubbles were slowly injected into the microbubbles by intravenous infusion of microbubbles (0.
  • Level 0 refers to a complete hemostasis
  • Level 1 refers to slow oozing
  • Level 2 refers to active bleeding of light::
  • Level 3 refers to bleeding visible to the naked eye
  • Level 4 refers to significant bleeding.
  • Three rabbits were randomly selected from each group, divided into two groups: one group obtained the irradiated target area liver tissue and the uninjured liver tissue immediately after laparotomy treatment, and the other group obtained the irradiated target area liver tissue after 48 hours of treatment. And did not damage the liver tissue.
  • Light microscopy observation placed in 10% formalin fixed, paraffin-embedded sections, HE staining; electron microscopic observation, after irradiation, the target area and undamaged liver tissue samples were obtained, fixed in 2. 5 % glutaraldehyde liquid for 24h Go to the school's electron microscope room.
  • Contrast-enhanced ultrasonography revealed that there was no change in liver perfusion in the control group with hepatic parenchymal blood flow in the ultrasound microbubble group. (image 3 ).
  • HE sections showed normal structure of the liver and clear hepatic sinus (Fig. 4A - 4C).
  • Pathological examination revealed an acute hemostatic mechanism, and we found two important lesions in histological observation. The first is the turbid swelling of hepatocytes in the ultrasound ⁇ :bubble specimens, which show a typical balloon-like change and squeeze the hepatic sinus gap (Fig. 4D-41). Hepatocyte swelling around the incision forced the hepatic sinus to almost disappear (Fig. 4F).
  • the second lesion was a significant portal vein hemorrhage with an irregular annular, cuff-like hematoma around the portal vein (Fig. 4D - 4E), but the portal vein wall was intact.
  • the liver tissues of 11 animals showed necrosis in the white-yellow region.

Abstract

微泡联合超声空化的止血用途，主要用于腹部实质性脏器创伤后出血的止血。血液循环中微泡超声造影剂在一种特征性脉冲式超声激励下，发生一定强度的超声空化，破坏局部组织的微血管，形成围绕血管的出血、血肿和血栓等急性病变，压迫并阻断局部肝脏血液循环，继而产生对肝创伤后出血的止血治疗作用。微泡经静脉注射进入人体血液循环；超声发射脉冲的峰值负压在1-9兆帕（MPa)、脉冲宽度超过20个周期、低占空比和低声强；该治疗在肝脏的微小血管腔内发生高强度的超声空化，并产生强烈的非热效应、机械破坏效应，可以导致肝组织的微小血管破裂损伤、出血、血肿和血栓形成、以及肝细胞浑浊肿胀等病理改变，对局部组织的血液循环产生暂时性的阻断作用，从而进一步产生止血治疗作用。

Description

泡联合超声空化的肝创伤止血用途 技术领域
本发明涉及^：泡在超声激励下的止血治疗新用途， 可以应用于肝实质 性脏器的创伤后止血治疗， 属于非创伤性超声治疗的技术领域。
技术背景
肝脏是腹部创伤中最容易受到损伤的器官， 其创伤后出血是腹部创伤 后最常见并发症， 导致的出血性休克是死亡的主要原因。 肝的血供丰富且 质地柔软的实质性器官， 肝中破裂出血和慢性渗血中约 50 %最后仍然需要 手术止血[1—2] , 复杂的肝创伤不易手术修补缝合， 而且手术创伤性大 [3]。 目 前， 尽管目前已经发展出超声造影引导下止血剂注射、 微波或射频热凝固 止血、 高强度聚焦超声术中止血、 介入性血管栓塞等方法， 但是以上止血 方法可能存在止血范围局限、 设备复杂、 需要精确影像导航、 耗时长等局 限性， 这些治疗手段往往不能满足患者的治疗要求， 故寻找一种非创伤性 的腹部创伤后止血方法十分必要。
高强度聚焦超声 （H igh intens i ty focused ul t rasound, HIFU )利用 超声的热效应进行肝脏创伤止血。 但是， 目前看来 HIFU肝脏止血需要在开 腹直视下的进行， 而深部出血则需要准确的影像引导聚焦超声能量。 该方 法需要较大的超声设备， 手术过程中均需要麻醉， 操作复杂。 如果不开腹 在体外实施， 则存在肋骨或者肠道等声阻抗界面阻挡、 热效应聚集差、 传 导不均匀等情况， 可能烧伤皮肤、 肋骨及肠道等周围重要器官， 产生严重 的副作用， 故体外治疗试验从未实施过。 其止血作用主要限于肝表面， 治 疗时间偏长，在治疗中受到很大程度的限制， 目前尚未在临床推广应用 [4_7]。
超声空化效应 ( cav i ta t ion ), 注： 空化效应是指液体中^:气泡在超声 作用下产生震荡、 膨胀、 收缩以及内爆等一系列动力学过程， 伴随瞬态高 温、 高压、 冲击波、 放电和微射流等多种能量释放行为） [8]是热效应之外另 一种超声的主要物理效应。
近年来， 泡超声造影剂增强的超声空化效应在非创伤性超声治疗领域 中的基础研究十分活跃， 但是大多数相关研究主要涉及微泡作为载体进行 药物释放和基因转染， 对其空化本身的机械性损伤效应研究较少。 生物体 内的声空化效应的发生和强度影响因素较多， 但最重要的因素是超声峰值 负压强度， 一般在峰值负压大于 0. 6MPa时就会发生显著的空化效应。 欧美 仅有 Hwang、 Ca in, Mi l ler和刘政等少数几个课题组进行过该领域的探讨 "― 13] , 他们的研究仅涉及到正常血管壁的空化物理损伤和离体组织标本的超 声空化消融， 釆用的治疗方法也不尽相同。 Hwang等釆用经静脉注射^：泡联 合聚焦超声空化损伤实现了对兔耳缘静脉血管内皮的损伤， 但是需要局部 管腔内注射小剂量凝血酶， 才可能造成血管栓塞； Ca in[ "的空化研究中没有 釆用静脉注射微泡， 而是利用超声激发生物体内自身潜在的微泡发生空化 进行组织消融， 釆用的是离体正常心肌等组织作为研究对象； Mi l ler等主 要研究微泡诱导的超声空化对于正常心肌或者肾脏等器官微血管的空化损 伤副作用， 他们的空化研究中均没有釆用肿瘤新生血管作为消融破坏对象。 国内吴巍等 [14— 15]釆用的是低频 ( 20-50KHz )低功率 ( 1-l OOW/cm2 )超声联合 微泡进行微血管栓塞， 不仅与本项目要求的 300KHZ-3. 0MHz、峰值负压大于 2. OMPa的脉冲式超声完全不同， 而且该研究文献中实验结果， 经反复重复 无栓塞阻断^：血管效果。 诊断超声虽然峰值负压也可以达到 2MPa以上， 但 是其脉冲宽度极短（约为 1个周期）， 不能诱导高强度的超声空化效应。 发明内容
本发明提出一种微泡在制备联合超声产生空化作为肝创伤出血症止血 药物的用途， 是一种新型非创伤性的超声空化止血用途。
所述微泡是一种在循环中在一定能量的超声脉冲激励下能够产生高强 度声空化的微泡； 所述空化可以机械性破坏局部肝脏的微小血管， 形成血 管周围出血、 血肿和血管内血栓； 空化效应产生的声孔效应还导致肝细胞 肿胀， 挤压肝窦， 导致局部肝组织暂时性的 循环阻断， 从而在肝创伤时 产生止血。
上述微泡选自超声造影剂微泡或者治疗型微泡， 选自声诺维 ( SonoVue )、 Def ini ty 、 O t i son™ 、 Sonazoid™ 、 Imagent、 全氟丙坑 人血白蛋白微球和全氟显； 微泡釆用经外周静脉注射方式进入人体， 微泡 注射液使用剂量在但不限于每公斤体重 0. 01—1. 0毫升范围。
上述的肝创伤后出血症， 是指肝脏在各种创伤情况下产生的出血现象， 包括战伤及腹部各种创伤后常见的并发出血症。
上述超声能量为脉冲式或者间歇脉冲式超声发射， 具有较高的峰值声 压和较低的发射占空比。
上述超声能量形式： 其发射的频率范围在 300千赫兹 -3. 0兆赫兹； 包 括峰值正压和峰值负压的超声峰值声压在 1-9 兆帕； 每个超声脉冲宽度在 20个周期以上； 其每次脉冲式超声发射治疗总时间 30秒以上； 脉冲式超声 发生可以是连续式， 也可以呈间歇式， 间歇或暂停时间为 0-20秒不等； 超 声发射占空比变化在 0. 01-10%之间。
上述高强度声空化是指微泡在权利要求 5 中所述的超声能量形式照射 激发情况下所发生的强烈声空化效应。
实现上述发射超声的治疗装置是各种形式的平面式发射或者聚焦式发 射的超声仪。
所述的^:泡增强的超声空化止血治疗作用， 在联合静脉使用凝血酶原 复合物或者纤维蛋白原时， 其止血作用可以得到进一步增强。
微泡的超声造影增强作用已经在临床诊断中被批准， 在本发明中属于 新的治疗用途。 在本发明设计的超声能量靶向照射作用下， 经静脉进入血 液循环的微泡会发生高强度的声空化效应， 首先可以造成显著的肝微小血 管机械性破坏、 出血和血肿形成， 进而产生血液循环的暂时 （血流灌注） 阻断效应， 在此基础上， 阻断了出血的来源而止血。 其特点是阻断血流灌 注不论是表面或者深层组织。 本发明的超声能量是指一种脉冲式或者间歇 脉冲式、 较高峰值声压、 较低的发射占空比的超声能量形式， 其发射的频 率范围在 300KHZ-3. OMHz; 超声发射可以呈连续脉冲式或者间歇脉冲式， 间 歇（暂停）时间从 2-20秒不等； 超声峰值负压在 2-10MPa之间； 超声发射 的每次脉冲宽度在 20 个周期以上； 超声发射占空比多变化在 0. 01-10%之 间；因而平均声强（ ISPTA )多低于 2W/cm2,热效应微小。每次治疗时间在 30-600 秒之间。 例如： 本研究使用的声学参数为超声发射频率 831KHz , 峰值声压 4. 47MPa , 脉冲宽度 400个周期， 脉冲重复频率 9Hz , 间歇脉冲式超声发射 (发射 6秒 /间歇 6秒）， 实际占空比 0. 22% ,平均声强仅 ( ISPTA ) 0. 4 W/cm2 。 附图说明
图 1为止血过程的示意图；
图中 A:以尺子刻度为标， 用手术刀切割一个 2cm的切口。 B: 收集 10 秒出血量， 计算初始出血速度。 C: 治疗仪探头覆盖切割伤伤口， 进行肝脏 止血治疗。 D: 超声治疗组治疗后伤口出血止血。
图 2为视觉评估（A )及总出血量（B )在超声微泡组、 单纯超声组、 假 照组之间的比较图；
图 3. 超声下观察超声^:泡组及假照组肝脏（A D) , 治疗前造影图像下 显示两组肝实质充盈良好（B E) , 治疗后假照组造影前后无差别（B C) ,微泡 超声组止血治疗后出现超声造影的充盈缺损（E F)；
图 4.止血后的肝组织结构示意图。
具体实施方式
(1)超声治疗仪声学测量
超声治疗仪器的其声学参数包括超声频率、 峰值负压、 脉冲宽度、 平 均声强 （I™)和占空比等， 一律以水听器法测量结果为准。 超声治疗仪信 号发出后，接收信号由距发射换能器 3cm处的针状水听器（NTR1000 , 美国） 接收， 经前置放大器（NTR, 美国）放大后由数字示波器（Ag i lent 55310, 美国）计算机釆样分析。 测量结果，
(2)肝脏创伤后止血实验
目的： 建立兔肝脏切割伤出血模型， 利用超声联合微泡空化效应进行 肝脏止血。
材料与方法：
(一）模型制备
健康新西兰大白兔 20只， 雌雄不限， 体重在 2000-2225g。 新西兰兔 以速眠新 II 0. 2mg/kg麻醉后，仰卧固定于自制木板上，建立耳缘静脉通道， 2%戊巴比妥钠 （ 0. 2ml /kg )经耳缘静脉注射麻醉后， 剑突到肋弓下缘腹部 备皮， 按外科无菌程序消毒术区皮肤， 常规消毒铺洞巾， 无菌条件下开腹， 剑突下 1 cm 处沿腹白线作长 1 ~ 2 cm 的正中切口，逐层开腹， 经手术暴露 肝脏后轻轻将肝左中叶及右肝牵出腹腔， 以生理盐水浸润纱布固定。 以手 术刀片切 2 X 0. 5cm大小伤口（图 2 A) , 造成肝脏切割伤出血模型（图 2 B )。
(二） 实验分组
随机分为三组：即超声微泡组（ n=8 )、单纯超声组（ n=6 )、假照组（η=6) , 超声微泡组釆用超声照射联合经静脉微泡注射； 单纯超声组仅有超声照射； 假照组仅施予超声治疗头假照。
(三）超声造影检查
各组创伤前、 治疗后超声造影， 分析各时间点为 0、 10min、 48h (共 3个时间点）超声造影灌注峰值灰阶变化及视觉评分方法分析微泡增强的超 声空化阻断兔肝实质血流的持续时间， 了解肝实质血流灌注的恢复情况（图 3 )。
(四）创伤后初始出血速度的计算
初始出血速度 ( ml /min )是纱布吸取切口造成后的出血量除以 10s时 间所得到的比值。 取无菌纱布称原始重量， 重量精确到 0. Olg , 切口造成后 立即按秒表计时， 同时纱布吸血， 10s 后纱布称重减去纱布原始重量得到 10s出血量。
(五）兔创伤后肝脏超声空化治疗
超声造影釆用编码激励谐波成像， 机械指数 0. 13 , 造影团注 "脂氟 显" 微泡 0. 01 ml /kg , 图像深度、 增益在实验中保持不变。 按设定时间和 超声定位方向辐照切割伤区肝脏， 超声 ^：泡组釆用超声照射联合经静脉 ： 泡注射； 单纯超声组仅有超声照射； 假照组仅施予超声治疗头假照。 超声 微泡组在治疗时经静脉通道緩慢推注微泡緩慢推注微泡（ 0. 1 ml /kg , 以生 理盐水稀释至 3ml )同时用超声治疗头垂直轻压辐照切割伤区肝脏 2min (图 1C ); 单纯超声组用 3 ml 生理盐水代替^:泡， 同时用超声治疗探头垂直轻 压辐照肝脏 2min; 假照组治疗头假照 2min, 用相同剂量的生理盐水替代 ： 泡。
(六）视觉评分
止血治疗后立即进行止血效果的视觉评分， 分为以下等级：
0级指一个完全止血；
1级指緩慢渗血；
2级指轻^:的活动性出血；
3级指肉眼可见的出血；
4级指显著的大出血。
(七）病理学检查
每组随机选取三只实验兔， 分两组： 一组为开腹治疗后立即获取辐照 靶区肝组织及未损伤肝组织， 一组为治疗后关腹 48h后获取辐照靶区肝组 织及未损伤肝组织。 光镜观察， 置于 10%甲醛液中固定， 石蜡包埋切片， HE 染色； 电镜观察， 获取辐照后靶区及未损伤肝组织样本， 于 2. 5 %戊二醛液 固定 24h后送往学校电镜室。
结果：
(―) 初始出血速度及出血重量 所有的肝切割伤对肝脏造成显著灌注出血（图 2 B )。 初始出血速度 在任何两组之间都没有显著差异。 治疗后， 超声微泡组视觉评分在 0-1级， 与对照组存在显著差异。 治疗 2分钟和釆血 8分钟共 10分钟的总出血量， 也表明超声 ^：泡组与对照组存在显著差异。
(二） 超声造影检查
超声造影发现， 超声微泡组治疗区域肝实质血流出现灌注缺损两个 对照组的治疗肝脏灌注基本没有改变。 （图 3 )。
(三） 病理检查
对照组 HE切片显示肝组织为正常结构， 可见清晰肝窦 （图 4A - 4C )。 病理检查显示急性止血机制， 我们在组织学观察发现两个重要病变。 第一 个是超声^:泡组标本切片肝细胞的混浊肿胀， 肝细胞表现出典型的气球样 变， 挤压肝窦间隙 （图 4D - 41 )。 切口周围肝细胞肿胀压迫肝窦几乎消失 (图 4F )。 第二个病变是显著的门静脉区出血，在门静脉周边形成一个不规 则的环形、 袖套状血肿（图 4D - 4E ), 但门静脉壁完整未受到损伤。 治疗 后 48个小时， 11只动物的肝组织呈白黄色区域坏死。
表 1. 各实验组在初始出血速度、视觉出血评分和治疗后出血量方面 的比较
初始出血速
平均视觉评 治疗后 10分钟 组 别 度
分 出血量（克）
(克 /秒）
超声微泡 0. 38 ±
0. 10 ± 0· 08 2. 21 ± 1. 26* 组 0. 52*
超声组 0. 12 ± 0· 04 3. 67 士 0· 52 14. 63 ± 7. 24 微泡组 0. 12 ± 0· 06 3. 83 ± 0· 41 11. 68 ± 3· 77 注： P <0. 01 *表示超声^:泡组与对照组相比， 存在显著差异。
结论：
^：泡联合低声强的超声为肝脏创伤出血症提供了一个新的止血用途。 参考文献
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Claims

权利要求
1. 微泡在制备联合超声产生空化作为肝创伤出血症止血药物的用途， 其特征是， 所述微泡是一种在循环中在一定能量的超声脉冲激励下能够产生高强度声空化的微 泡； 所述空化可以机械性破坏局部肝脏的微小血管， 形成血管周围出血、 血肿和血管 内血栓； 空化效应产生的声孔效应还导致肝细胞肿胀， 挤压肝窦， 导致局部肝组织暂 时性的微循环阻断， 从而在肝创伤时产生止血。
2. 权利要求 1 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途， 其特征是， 微泡是超声造影剂微泡或者治疗型微泡， 选自声诺维 （SonoVue )、 Def ini ty 、 Opt i son 、 Sonazo id 、 Imagent . 全氟丙垸人血白蛋白微球和全氟显； 微泡釆用经外周静脉注射方式进入人体， 微泡注射液使用剂量在但不限于每公斤体重 0. 01—1. 0毫升范围。
3. 权利要求 1 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途， 其特征是， 所述的肝创伤后出血症， 是指肝脏在各种创伤情况下产生的出血现 象， 包括战伤及腹部各种创伤后常见的并发出血症。
4. 权利要求 1 所述微泡在联合超声产生空化作为肝创伤后出血症的止血药物的 用途， 其特征是， 所述的超声能量为脉冲式或者间歇脉冲式超声发射， 具有较高的峰 值声压和较低的发射占空比。
5. 权利要求 4 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途，其特征是，所述的超声能量形式： 其发射的频率范围在 300千赫兹 -3. 0兆赫兹； 包括峰值正压和峰值负压的超声峰值声压在 1-9兆帕； 每个超声脉冲宽度在 20个周 期以上； 其每次脉冲式超声发射治疗总时间 30 秒以上； 脉冲式超声发生可以是连续 式，也可以呈间歇式，间歇或暂停时间为 0-20秒不等；超声发射占空比变化在 0. 01-10% 之间。
6. 权利要求 1 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途， 其特征是， 所述的高强度声空化是指微泡在权利要求 5中所述的超声能量形式 照射激发情况下所发生的强烈声空化效应。
7. 权利要求 5 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途， 其特征是， 发射超声的治疗装置是各种形式的平面式发射或者聚焦式发射的超 声仪。
8. 权利要求 1 所述微泡在制备联合超声产生空化作为肝创伤出血症止血药物的 用途， 其特征是， 所述的微泡增强的超声空化止血治疗作用， 在联合静脉使用凝血酶 原复合物或者纤维蛋白原时， 其止血作用可以得到进一步增强。
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