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。     

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。     

MRI引导聚焦超声联合微泡开放血脑屏障的时效关系

目的研究在MRI引导下聚焦超声联合微泡靶向开放血脑屏障（BBB）的时效关系。 方法采用1.5 T的MRI超声治疗系统联合微泡辐照靶点,2 h后增强T1相扫描,测定靶点信号强度值与病理组织学和MR图像结果比较。 结果当声裂10 s和14 s时,靶点信号强度值出现最高值（P〈0.05）,而脑组织损伤不明显;16 s组或更长时间,信号强度值不再增加或出现下降,组织学检查发现有不同程度坏死、红细胞渗出等病理改变。 结论MRI引导聚焦超声联合微泡可靶向开放局部血脑屏障;T1相信号强度值监测BBB的通透性。     

低频聚焦超声联合微泡开放血脑屏障的研究进展

血脑屏障(blood brain barrier,BBB)作为大脑的保护性屏障,有效的维持着中枢神经系统(centre nerve system,CNS)内环境的稳定,但同时也是大多数药物或基因等进入脑内发挥作用的主要障碍,影响了颅内疾病的有效治疗。因此如何有效的开放血脑屏障是科学家们历年来一直关注研究的热点。聚焦超声具有方向     

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。     

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。     

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.     

超声联合微泡开放血脑屏障及其安全性的研究

血脑屏障（blood-brain barrier，BBB）是指脑毛细血管阻止某些物质（多是有害的）进入脑循环血的结构，这种结构可使脑组织少受甚至不受循环血液中有害物质的损害，从而保持脑组织内环境的基本稳定，对维持中枢神经系统正常生理状态起着重要作用。但由于血脑屏障的存在，其在阻止有害物质进入脑组织的同时，也阻碍了有效治疗药物进入中枢神经系统，     

Focused Ultrasound and Microbubble Treatment Increases Delivery of Transferrin Receptor-Targeting Liposomes to the Brain

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain.     

Multi-Modality Safety Assessment of Blood-Brain Barrier Opening Using Focused Ultrasound and Definity Microbubbles: A Short-Term Study

As a potentially viable method of brain drug delivery, the safety profile of blood-brain barrier (BBB) opening using focused ultrasound (FUS) and ultrasound contrast agents (UCA) needs to be established. In this study, we provide a short-term (30-min or 5-h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity microbubbles (0.05 μL/kg) and sonicated under the following parameters: frequency of 1.525 MHz, pulse length of 20 ms, pulse repetition frequency of 10 Hz, peak rarefactional acoustic pressures of 0.15–0.98 MPa and two 30-s sonication intervals with an intermittent 30-s delay. The BBB opening threshold was found to be 0.15–0.3 MPa based on fluorescence and magnetic resonance imaging of systemically injected tracers. Analysis of three histological measures in hematoxylin and eosin–stained sections revealed the safest acoustic pressure to be within the range of 0.3–0.46 MPa in all examined time periods post sonication. Across different pressure amplitudes, only the samples 30 min post opening showed significant difference (p < 0.05) in the average number of distinct damaged sites, microvacuolated sites, dark neurons and sites with extravasated erythrocytes. Enhanced fluorescence around severed microvessels was also noted and found to be associated with the largest tissue effects, whereas mildly diffuse BBB opening with uniform fluorescence in the parenchyma was associated with no or mild tissue injury. Region-specific areas of the sonicated brain (thalamus, hippocampal fissure, dentate gyrus and CA3 area of hippocampus) exhibited variation in fluorescence intensity based on the position, orientation and size of affected vessels. The results of this short-term histological analysis demonstrated the feasibility of a safe FUS-UCA–induced BBB opening under a specific set of sonication parameters and provided new insights on the mechanism of BBB opening. (E-mail: ek2191@columbia.edu)     

Focused Ultrasound Microbubble Destruction‐Mediated Changes in Blood‐Brain Barrier Permeability Assessed by Contrast‐Enhanced Magnetic Resonance Imaging

Objective. The purpose of this study was to use enhanced magnetic resonance imaging (MRI) to evaluate the changes of blood‐brain barrier (BBB) permeability in target and nontarget areas of rabbit brains after BBB disruption induced by focused ultrasound‐mediated microbubble destruction. Methods. Focused ultrasound (1.1 MHz) in combination with a sulfur hexafluoride microbubble contrast agent was applied at 2 or 3 target locations in 1 hemisphere of 29 rabbit brains to induce BBB disruption. The opposite side was used as a control, and a normal group contained another 14 rabbits that did not undergo sonication. The MRI signal intensity enhancement in the target locations was detected to evaluate gadolinium (Ga) retention after sonication, and extravasation of Evans blue (EB) dye was detected to evaluate the BBB disruption quantitatively at different times after sonication (0.5, 2, 4, 6, 8, and 24 hours and 1 week). Results. Compared with the control group, Ga retention, changes in EB content, and extravasation in the cerebral cortex of the sonicated group peaked at 2 hours (P < .01) and decreased to the normal level 8 hours after sonication (P < .01). There was no visual evidence of injury or hemorrhage within the brain parenchyma of all of the rabbits' treated hemispheres. Conclusions. Magnetic resonance imaging–guided focused ultrasound can disrupt the BBB reversibly and can allow targeted delivery of some molecules that normally cannot cross the BBB to locations in the brain. Changes in BBB permeability develop within minutes after sonication as a result of a combination of factors. The BBB has a self‐repairing characteristic, which is activated after ultrasound sonication. This may offer an improvement in future clinical applications for central nervous system diseases.     

两种开放血脑屏障方法的比较

目的 对比聚焦超声联合微泡和高渗性甘露醇2种方法对开放大鼠血脑屏障(BBB)的影响.方法 将115只SD大鼠分为聚焦超声联合微泡组(A组)50只、甘露醇组(B组)50只、假手术组(C组)15只.拟开放BBB后用磁共振增强扫描观察其开放时间特点,用荧光显微镜观察伊文氏蓝和右旋糖酐脑内空间分布差异,荧光分光光度法进行定量分析.结果 A组BBB局部开放,B组弥散广泛开放;A组于0 min核磁信号增强达峰值,B组于5 min信号增强达峰值.10 kDa的右旋糖酐荧光信号较40 kDa的右旋糖酐强且弥散.A组脑组织内伊文氏蓝、10 kDa的右旋糖酐和40 kDa的右旋糖酐的含量均明显大于B组(P＜0.01).结论 聚焦超声联合微泡开放BBB的方法较甘露醇法开放BBB程度更大,开放范围局限,靶向性、可控性更好.     

脑超声造影中超声造影剂剂量对血脑屏障通透性的影响

目的探讨不同剂量超声造影剂在超声造影中对血脑屏障通透性的影响。方法60只清洁级SD大鼠,给予相同机械指数的超声进行辐照,并经尾静脉注射不同剂量的“脂氟显”超声造影剂,观察超声照射后血脑屏障通透性的变化。结果在造影剂剂量为50μl/kg时,血脑屏障通透性即与对照组相比产生显著性统计学差异,且随着超声造影剂剂量的增加其引起血脑屏障通透性也随之增加。结论在相同能量强度超声场中,超声造影剂微泡数目是引起不同生物学效应的重要因素。     

电镜硝酸镧示踪超声微泡造影剂开放血脑屏障

目的 研究超声波破坏微泡造影剂对大鼠血脑屏障通透性的影响。 方法 用电镜硝酸镧示踪法观察超声波破坏微泡造影剂对大鼠血脑屏障通透性的变化。 结果 超声波照射后即刻，即可见镧颗粒通过毛细血管内皮细胞及细胞间的紧密连接进入组织间隙，血脑屏障开放持续至6h，12h时已关闭。电镜下可以见到血管源性脑水肿，细胞器水肿不明显。 结论 超声波破坏微泡造影剂开放血脑屏障，为中枢神经系统疾病的治疗提供了一种无创、具靶向性的药物转运方法。     

聚焦超声治疗574例宫颈炎临床分析

目的：探讨聚焦超声治疗慢性宫颈炎的安全性和有效性。方法：回顾性分析2003年1月至2006年12月我院门诊诊断治疗的慢性宫颈炎患者,行聚焦超声治疗后并于3月内随访的574例患者,分析其安全性及有效性。结果：574例中,痊愈378例（65．9％）,显效155例（27．0％）,总有效率96．7％。治愈率与糜烂面积及深浅程度有关（P〈0．05）。超声治疗后部分患者出现阴道少量流液及血性分泌物。结论：聚焦超声应用于慢性宫颈炎的治疗安全有效,疗效确切,不良反应及并发症小,值得推广。