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。     

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.     

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。     

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。     

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.     

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。     

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     

低频诊断超声联合脂氟显微泡开放小鼠血脑屏障的可逆性研究

目的探讨低频诊断超声联合脂氟显微泡开放小鼠血脑屏障(blood-brain barrier,BBB)的可逆性及伊文思蓝(Evans blue,EB)透过率。方法健康C57BL/6小鼠48只,随机分为对照组6只和观察组42只。观察组再随机分为0、0.5、1、2、3、8、10 h亚组各6只,低频诊断超声辐照同时经尾静脉注射脂氟显微泡1 mL/kg,并分别于超声辐照0、0.5、1、2、3、8、10 h经尾静脉注射质量分数2%EB溶液50 mg/kg;对照组不进行低频诊断超声辐照,经尾静脉注射1 mL/kg生理盐水后注射质量分数2%EB溶液50 mg/kg。注射1 h后处死小鼠取脑组织,观察脑组织蓝染深度及面积,采用紫外分光光度法定量分析脑组织EB含量。结果对照组及观察组8、10 h亚组未见脑实质蓝染,观察组0、0.5、1、2、3 h亚组辐照区脑实质均有不同程度蓝染,且1 h时蓝染深度最深、面积最大;观察组0、0.5、1、2、3 h亚组脑组织EB含量[(45.43±2.89)、(50.94±1.25)、(79.79±1.12)、(51.55±1.20)、(31.60±1.77)μg/g]均高于对照组[(8.32±0.12)μg/g](P<0.05),8、10 h亚组脑组织EB含量[(8.34±0.09)、(8.31±0.07)μg/g]与对照组比较差异无统计学意义(P>0.05);观察组0、0.5、1 h亚组脑组织EB含量依次升高(P<0.05),1、2、3 h亚组脑组织EB含量依次降低(P<0.05)。结论低频诊断超声联合脂氟显微泡开放小鼠BBB呈动态可逆的过程,BBB在超声辐照后1 h开放程度最大,于超声辐射8 h恢复正常。     

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.     

MRI温度图监控聚焦超声开放家兔血脑屏障的作用

目的探讨MRI温度图监控聚焦超声波开放家兔血脑屏障（BBB）的可行性及安全性。方法20只家兔分3组，分空白对照组、超声组和超声＋微泡组，分别在1．1MHz聚焦超声波，10W功率下照射不同时间，从6～20s不等。照射过程中应用MRIGRE序列实施实时监测焦点温度变化图，照射后进行MRIT2扫描和T1造影增强扫描，病理学检查。结果平均温度44．6℃到48．8℃之间、峰值温度45．9℃～51．6℃之间，可见BBB开放，而脑组织未见明显坏死，而在此温度之上，则有明显的脑组织坏死。结论MRI实时温度图可以预测聚焦超声开放家兔BBB过程中的脑组织改变，并监控其安全范围。     

经颅脑超声造影对血脑屏障通透性的影响

目的探讨经颅脑超声造影检查中血脑屏障通透性改变后其自行恢复时间，以探讨脑超声造影检查对血脑屏障通透性改变是否为可逆性。方法70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，辅以机械指数为1．3的经颅脑超声造影检查，观察超声辐照后不同时间点血脑屏障通透性的变化。结果在造影检查后12h，血脑屏障通透性与对照组相比差异无统计学意义，且随着脑超声造影检查后时间的推移，血脑屏障通透性逐渐恢复。结论高机械指数的体表超声辅以高剂量超声造影剂在经颅脑超声造影中可导致血脑屏障通透性增加，但在造影检查后，这种改变可自行恢复。     

Quantitative evaluation of focused ultrasound with a contrast agent on blood-brain barrier disruption

The use of an ultrasound contrast agent (UCA) with focused ultrasound sonication has the potential to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations. This study investigated the effects of UCA dose and ultrasound pressure on BBB disruption. Sonications were applied at 1 MHz with a burst length of 10 ms, a 1% duty cycle and a repetition frequency of 1 Hz. The duration of the sonication was 30 s. In in vivo experiments, 16 male Wistar rats were sonicated in the presence of UCA at four doses (0, 30, 60 and 90 microL/kg). BBB integrity was evaluated by injecting Evans blue (EB) into the femoral vein of anesthetized rats. The relationship between UCA dose and the region of EB extravasation was evaluated at ultrasound pressures of 0.9 and 1.2 MPa. The BBB disruption, as quantified by the amount of EB extravasation, was significantly greater in rats injected with UCA at a dose of 60 or 90 microL/kg than at a dose of 0 or 30 microL/kg. The amount of EB extravasation increased monotonically with the quantity of UCA injected into the femoral vein before sonication. Furthermore, the BBB disruption could be enhanced in the focal region relative to the surrounding region with a higher dose of UCA (60 or 90 microL/kg). This study demonstrates that BBB disruption can be both increased and localized to the focal region by injecting an appropriate quantity of UCA before performing focused ultrasound sonications.     

MRI引导与监测聚焦超声靶向开放血脑屏障兔脑给药

目的 探讨MRI引导与监测聚焦超声靶向开放血脑屏障(BBB)兔脑给药的效果.方法 25只新西兰大白兔随机均分为5组(0 h,2 h,4 h,8 h,24 h组),MRI引导聚焦超声辐照各组兔左脑,取靶点组织作为辐照组,取右脑相应解剖组织为对照组,分别于辐照后不同时间点静注欧乃影和甲氨喋呤(MTX),行T1加权相增强扫描,通过比较辐照组与对照组MRI信号强度增强率观察靶点BBB开放情况,并以靶点伊文氏蓝(EB)染色结果检验其准确性;以高效液相色谱法测定、比较辐照组与对照组MTX的浓度,计算靶点脑组织信号强度增强率与MTX浓度之间的线性相关系数.结果 兔脑靶点BBB开放处T1WI表现为点状或斑片状异常高信号影,与辐照前MRI定位时预设靶点位置吻合良好,并与EB蓝染结果一致,同时靶点脑组织信号强度增强率与MTX浓度之间的相关系数为0.96(P<0.01),两者有良好的相关性.结论 MRI能够引导与监测聚焦超声靶向开放BBB脑内给药,有望成为临床个体化治疗中枢神经系统疾病的理想监测手段.     

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.     

Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications

The purpose of this study was to test the hypothesis that burst ultrasound in the presence of an ultrasound contrast agent can disrupt the blood-brain barrier (BBB) with acoustic parameters suitable for completely noninvasive exposure through the skull. The 10-ms exposures were targeted in the brains of 22 rabbits with a frequency of 690 kHz, a repetition frequency of 1 Hz, and peak rarefactional pressure amplitudes up to 3.1 MPa. The total exposure (sonication) time was 20 s. Prior to each sonication, a bolus of ultrasound contrast agent was injected intravenously. Contrast-enhanced MR images were obtained after the sonications to detect localized BBB disruption via local enhancement in the brain. Brain sections were stained with H&E, TUNEL, and vanadium acid fuchsin (VAF)-toluidine blue staining. In addition, horseradish peroxidase (HRP) was injected into four rabbits prior to sonications and transmission electron microscopy was performed. The MRI contrast enhancement demonstrated BBB disruption at pressure amplitudes starting at 0.4 MPa with approximately 50%; at 0.8 MPa, 90%; and at 1.4 MPa, 100% of the sonicated locations showed enhancement. The histology findings following 4 h survival indicated that brain tissue necrosis was induced in approximately 70-80% of the sonicated locations at a pressure amplitude level of 2.3 MPa or higher. At lower pressure amplitudes, however, small areas of erythrocyte extravasation were seen. The electron microscopy findings demonstrated HRP passage through vessel walls via both transendothelial and paraendothelial routes. These results demonstrate that completely noninvasive focal disruption of the BBB is possible.