低频超声联合微泡经颅开放血脑屏障初步研究

目的探讨低频超声联合微泡经颅靶向开放血脑屏障及在临床应用价值。方法①经静脉注射微泡后,用频率为43kHz、强度为2W/cm2,持续超声波辐射大鼠头部5min;②采用荧光显微镜伊文氏蓝(EB)、电镜镧示踪法以及透射电镜观察脑组织的超微结构变化。结果经颅超声波与微泡联合能短暂地促进伊文氏蓝和镧离子通过多种形式跨越血脑屏障。结论低频超声联合微泡能可逆的、靶向的、局部的开放血脑屏障,并为进一步研究药物进入脑实质内提供新的策略。     

低频聚焦超声联合微泡开放大鼠血脑屏障的时间参数对比研究

目的 探索低频聚焦超声联合微泡开放大鼠血脑屏障的辐照时长参数.方法 健康SD大鼠60只,随机分为对照组(A组)、超声组(B组)和超声微泡组,超声微泡组按辐照时间不同分为0.5min组(C1组)、1.0 min组(C2组)、1.5 min组(C3组)、3.0 min组(C4组);实验鼠经尾静脉注射自制微泡和伊文思蓝染料后,用频率为1 MHz,声强为4 W/cm2的低频聚焦超声以不同辐照时间辐照大鼠头部,采用组织学方法 观察大鼠血脑屏障的开放情况.结果 超声微泡组辐照时间0.5 min(C1组)未见脑组织蓝染,辐照时间1.0 min组(C2组)、1.5 min组(C3组)、3.0min (C4组)组均可见蓝染;HE染色检测3.0 min组(C4组)见血细胞.结论 低频聚焦超声联合微泡在频率为1 MHz、声强4 W/cm2、辐照时间1.0～1.5 min参数下可以局部开放血脑屏障.     

诊断超声联合微泡开放体外血脑屏障的机理研究

目的 建立体外血脑屏障(blood brain barrier,BBB) 模型,探讨诊断超声联合造影剂(微泡)可逆性开放血脑屏障的机理.方法 BALB/c小鼠脑微血管内皮细胞(brain microvascular endothelial cells,BMVEC) 建立BBB体外实验模型.选择青壮年标本颞骨片,大小1.5 cm×2.0 cm,置于细胞小室上,模拟经颅骨超声声窗条件,根据分组在模型中加入自制的脂膜氟烷超声造影剂(微泡),采用经头颅超声诊断仪辐照,辐照10 min.分为4组,每组4个样本:(1)对照组;(2)超声组;(3)微泡组;(4)超声联合微泡组.经上述处理后,光镜和电镜观察BBB细胞膜及超微结构改变,在不同的时间点检测跨内皮细胞电阻(transendothelial electrical resistance,TEER)和辣根过氧化物酶(horseradish peroxidase,HRP) 的通透性,探讨可逆开放BBB的机理.结果 光镜和扫描电镜显示实验后4组样本细胞表面无明显形态学改变和损伤,透射电镜证实超声联合微泡组辐照后细胞间紧密连接向桥粒连接过渡,超声组紧密连接减少,但没有明显连接分离现象; HRP通透率检测显示超声联合微泡组辐照后细胞通透率呈波浪形增加,18 h后完全恢复,超声组辐照后BBB通透率增加,于30 min内恢复,通透率最高时跨细胞电阻抗(TEER)降低至(179±8) Ω/cm2,随着HRP通透率的减低,TEER逐渐恢复;电镜、HRP通透率在微泡组和对照组没有明显改变.结论 微泡联合诊断超声波能通过短暂开放紧密连接,降低细胞间电阻,提高通透率,达到可逆性开放血脑屏障.     

低频超声破坏微泡造影剂开放血脑屏障的实验研究

目的探讨不同强度的低频超声经颅诱导微泡造影剂破坏对大鼠血脑屏障的影响。 方法股静脉注入微泡造影剂后，采用频率43kHz，声强分别为1．2．1．5．1．8．2．3w／cm。的连续超声波经大鼠颅骨照射3min，荧光显微镜观察伊文思兰的渗出。 结果注射微泡造影剂后，声强1．2w／cm^2时超声波即可以开放血脑屏障，随声强的增加脑组织损伤加重。 结论低频超声诱导微泡造影剂破坏可以靶向开放血脑屏障。     

诊断超声联合微泡靶向开放单侧大鼠血脑屏障的初步研究

目的初步探讨诊断超声靶向击破微泡开放大鼠单侧血脑屏障通透性的可行性、有效性及安性。方法25只健康SD大鼠,诊断超声经颞骨照射单侧大脑半球,对侧大脑半球作对照。对伊文思蓝外渗范围进行分级和萃取定量。透射电镜观察硝酸镧分布评价血脑屏障超微结构变化,并对双侧脑实质的病理损伤进行评价和分级。结果照射侧大脑半球可见以海马为中心的片状均匀伊文思蓝渗出,而对侧脑实质未见渗出;透射电镜照射侧大脑半球硝酸镧渗出血管并分布于神经纤维间隙,而对照侧未见硝酸镧外漏;超微结构显示少量神经元轻度肿胀。病理组织学观察照射侧大脑半球可见少量红细胞外漏,未见明显组织细胞损伤。结论诊断超声靶向击破微泡能安全有效靶向开放单侧大鼠血脑屏障且呈海马为中心分布,这对于颅内以海马为中心分布疾病的靶向性药物治疗具有潜在的临床价值。     

聚焦超声联合微泡开放血脑屏障的研究进展

血脑屏障可以阻止有害物质进入脑内,同时阻碍了药物治疗颅内疾病。如何安全、可逆地开放血脑屏障成为研究重点。聚焦超声联合微泡技术开放血脑屏障可能是基于超声的空化效应和声辐射力作用,其开放程度可在MRI的监控下准确评估,且微泡作为载体可运载辅助药物治疗颅内疾病,已在大量动物实验中获得初步成效。该技术具有靶向、无辐射和无毒性、不损害大脑组织及可重复性等优点,具有很大的发展前景。     

两种微泡造影剂开放大鼠血脑屏障的比较性研究

目的 探讨自制磷脂微泡和声诺维两种微泡对大鼠血脑屏障的影响.方法 将32只SD大鼠随机分为自制微泡即刻组、自制微泡24 h组、声诺维即刻组和声诺维24 h组,各组由尾静脉注射相应微泡后,在探头频率为1 MHz、声强4 W/cm2、辐照时间1.0 min参数下超声照射大鼠脑部,大体观察脑组织的蓝染程度及范围;伊文思蓝测定法观察大鼠血脑屏障通透性;光镜下观察脑组织、脑细胞和血脑屏障病理学改变.结果 声诺维24 h组大鼠脑组织蓝染程度及范围明显小于其他各组,脑组织通透性明显低于其他各组（P﹤0.05）,HE染色各组未见大鼠脑组织损伤及血细胞渗出.结论 自制磷脂微泡开放大鼠血脑屏障效果与声诺维相比无明显差异,且自制微泡开放血脑屏障的效果更为稳定.     

脑超声造影中超声强度对血脑屏障通透性的影响

目的探讨不同机械指数的诊断性超声在超声造影中对血脑屏障通透性的影响,以了解脑超声造影检查中超声强度的安全应用范围。方法50只清洁级SD大鼠,经尾静脉注射剂量为1ml/kg的“脂氟显”超声造影剂,辅以不同机械指数的超声进行辐照,观察超声照射后血脑屏障通透性的变化。结果在MI等于0.4时,血脑屏障通透性与对照组相比无统计学差异,当MI≥0.8时,血脑屏障通透性增加,且随着超声能量的进一步提高血脑屏障的通透性增加。结论高机械指数的体表超声在超声造影中可导致血脑屏障通透性增加,但应用适当强度的超声在进行大脑超声造影时仍是安全的。     

聚焦超声联合微泡开放血脑屏障对小鼠紧密连接蛋白的影响

摘要目的：探讨聚焦超声（focused ultrasound, FUS）联合微泡开放血脑屏障（blood-brain barrier, BBB）的安全性,并通过对FUS辐照后紧密连接蛋白的检测探究FUS开放BBB的可能机制。方法：健康昆明小鼠70只,随机分为对照组、FUS联合微泡开放BBB组,并进一步分为FUS开放BBB1h、4h、24h组。采用Garcia量表评估小鼠的神经行为学变化;HE染色观察超声辐照脑区的出血情况;ELISA动态检测炎性因子：肿瘤坏死因子α（tumor necrosis factor alpha,TNF-α）、白细胞介素1β（interleukin 1 beta,IL-1β）、白细胞介素6（interleukin 6,IL-6）的表达水平。经尾静脉注射伊文思蓝（evans blue, EB）动态观察不同分组小鼠脑内EB的渗透情况。Western Blot检测水通道蛋白4（aquaporin-4,AQP4）表达量的变化分析脑组织的水肿情况;分别检测不同时段紧密连接蛋白occludin、claudin-5、紧密连接蛋白1（zonula occludens-1,ZO-1）的表达水平评估超声对紧密连接蛋白的影响。结果：FUS诱导BBB开放后,小鼠不同时段的神经行为学评分均与对照组无明显差异（P>0.05）;HE染色显示FUS辐照后小鼠脑实质内无明显的红细胞渗出,炎性因子TNF-α、IL-1β、IL-6的表达水平在BBB开放后的1h、4h、24h均与对照组无明显差异（P>0.05）。在EB渗透实验中,1h组小鼠脑实质内有EB渗透,而其他分组均未观察到EB。不同时段AQP4蛋白表达量与对照组相比无显著性差异（P>0.05）。FUS辐照后1h,occludin（P=0.034）、claudin-5（P<0.001）、ZO-1（P=0.002）蛋白表达水平显著降低;4h后ZO-1、occludin蛋白水平逐渐上升至对照组水平（P>0.05）。而claudin-5蛋白表达量在24h仍低于对照组(P=0.002)。结论：FUS可以安全有效的开放BBB,其机制可能是通过作用于紧密连接蛋白ZO-1、occludin、claudin-5实现的。     

Noninvasive, transcranial and localized opening of the blood-brain barrier using focused ultrasound in mice

The feasibility of blood-brain barrier (BBB) opening in the hippocampus of wild-type mice using focused ultrasound (FUS) through the intact skull and skin was investigated. Needle hydrophone measurements through ex vivo skulls revealed minimal attenuation ( approximately 18% of the pressure amplitude), a well-focused beam pattern and minute focus displacement through the parietal bone. In experiments in vivo, the brains of three mice were sonicated transcranially. Pulsed ultrasound sonications at 1.5 MHz and acoustic pressures ranging from 0.8 to 2.7 MPa were used at 20% duty cycle. Before sonication, a bolus of 10 microL of an ultrasound contrast agents (Optison) was injected intravenously. Contrast-enhanced high-resolution magnetic resonance imaging (9.4 T) revealed BBB opening and allowed for the monitoring of the slow permeation of gadolinium in the hippocampus. The region of the brain where BBB opening occurred increased with the pressure amplitude. These findings thus demonstrated the feasibility of locally opening the BBB in mice using FUS through intact skull and skin and serve as the first step in determining and assessing feasibility of drug delivery to specific regions in the mouse brain using FUS.     

低频超声联合微泡促进多发性大动脉炎血管平滑肌细胞凋亡的生物学效应

目的:探讨超声联合微泡技术影响多发性大动脉炎(Takayasu arteritis, TA)血管平滑肌细胞的变化,为该类疾病的预防和治疗提供依据。方法:通过手术获取TA增生进展期、炎症硬化期及经皮血管腔内成形术(percutaneous transluminal angioplasty, PTA)后再狭窄期血管平滑肌细胞进行体外培养,建立偶联抗体的靶向超声微泡剂,采用20 kHz低频超声辐照150 s后培育24 h进行研究观察。结果:通过超声微泡处理后3组细胞存活率有明显差异(P0.01);相较其他两组,PTA后再狭窄期血管平滑肌细胞反应更敏感(P0.05),其培养基活性氧(ROS)明显上升(P0.01),而超氧化物歧化酶(SOD)活性明显降低(P0.01),细胞凋亡水平明显增加(P0.01)。结论:超声微泡联合处理可能对PTA后再狭窄期的治疗更有效果,超声微泡对TA血管平滑肌细胞具有明显的抑制与促凋亡的生物学效应。     

伊文思蓝定量评估超声造影对鼠胎盘屏障通透性影响的实验研究

目的探讨不同机械指数诊断性超声造影对胎盘屏障通透性的影响。方法怀孕14～16d（孕中期）清洁级SD鼠60只,经尾静脉注射剂量为1ml/kg的“声诺维”超声造影剂,在不同机械指数下（MI：0.13,1.0,1.4）超声辐照孕鼠子宫,辐照时间（连续5min,间歇10s）,分光光度法测定胎盘及胎鼠组织内伊文思蓝（EB）含量。结果不同机械指数下的超声造影,胎鼠组织内EB含量与对照组相比差异无统计学意义。结论高机械指数及低机械指数诊断超声造影均不会导致孕鼠胎盘屏障通透性增加。     

Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles

Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings--fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma.     

Treatment of deeply located acute intravascular thrombi with therapeutic ultrasound guided by diagnostic ultrasound and intravenous microbubbles.

OBJECTIVE: We sought to determine the added value of simultaneous imaging of intravenously infused microbubbles that are being used to dissolve an intravascular thrombus with therapeutic ultrasound (TUS). METHODS: In a chronic canine arteriovenous graft occluded by a thrombus, TUS (1 MHz) was applied through a 6-cm-thick tissue-mimicking phantom (measured mean +/- SD peak negative pressure through the phantom, 958 +/- 104 kPa) during an intravenous infusion of either saline (n = 6 occlusions) or lipid-encapsulated microbubbles (ImaRx Therapeutics, Inc, Tucson, AZ). Therapeutic ultrasound was intermittently applied during the microbubble infusion either at set time intervals (n = 6 occlusions) or when simultaneous diagnostic ultrasound (DUS) indicated a sustained presence of microbubbles (n = 12 occlusions). Success was defined as return of rapid flow within the graft (grade 3 flow). RESULTS: Diagnostic ultrasound showed microbubbles moving through small channels within the thrombus before angiographic evidence of flow in the graft. This guided the timing of TUS application better than using set time intervals. Angiographic clearance of the thrombus and restoration of grade 3 flow at 45 minutes of treatment were seen in 33% of deeply located thrombosed grafts treated with TUS at set time intervals and 92% of grafts treated with TUS guided by DUS (P < .001 compared with set time intervals). CONCLUSIONS: The use of TUS with intravenous microbubbles has a high success rate in recanalizing deeply located thrombosed arteriovenous grafts when performed with DUS guidance.