在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法
Summary
用微泡剂聚焦超声波可以聚焦和瞬时打开血脑屏障。这项技术已被用于通过血脑屏障提供广泛的制剂。本文提供了一个详细的协议，本地化交付给啮齿动物的大脑有或没有MRI指导。
Abstract
立体税手术是局部药物和基因输送到啮齿动物大脑的黄金标准。与系统传递相比，这项技术有许多优点，包括精确定位到目标大脑区域和减少目标外的副作用。然而，立体轴手术是高度侵入性的，它限制了其转化功效，需要很长的恢复时间，并在瞄准多个大脑区域时提供挑战。聚焦超声波 （FUS） 可与循环微泡结合使用，在毫米大小的区域瞬时打开血脑屏障 （BBB）。这允许系统交付的代理的颅内本地化，这些代理通常不能穿过 BBB。这项技术提供了立体轴手术的非侵入性替代方法。然而，由于获得设备和标准化方法的机会有限，该技术迄今尚未在神经科学实验室得到广泛采用。该协议的总体目标是为 FUS BBB 开启 （BBBO） 提供一种台面方法，这种方法经济实惠且可重复，因此很容易被任何实验室采用。
Introduction
尽管在基础神经科学方面有许多发现，但神经发育和神经退行性疾病的新疗法数量仍然相对有限。对神经紊乱中涉及的基因、分子和细胞回路的更深入的了解表明，用目前的技术3，人类无法实现有希望的治疗方法。有效的治疗往往受到大脑渗透和特定地点4，5，6，7，8的需要的限制。然而，现有的局部药物输送到特定大脑区域的方法（例如，通过注射或针管分娩）是侵入性的，需要在头骨9中打开。这种手术的侵入性防止了将局部分娩的常规使用到人脑中。此外，组织损伤和由此产生的炎症反应是无处不在的混淆的基本和前科研究，依靠颅内注射10。非侵入性地通过血脑屏障（BBB）输送制剂并将其靶向特定大脑区域的能力可能会对神经系统疾病的治疗产生巨大影响，同时为术前研究提供强大的研究工具。
在BBB上以最小的组织损伤进行定向传输的方法之一是颅内聚焦超声波（FUS）和微泡，以聚焦和瞬时打开BBB 11、12、13、14、15、16。FUS BBB 的开放最近因神经退行性疾病的治疗而备受关注， 中风和胶质瘤通过本地化治疗目标大脑区域，如神经营养因子17，18，19，基因疗法20，21，22，抗体23，神经递质24，和纳米粒子25，26，27，28，29。FUS BBB 具有广泛的应用范围和非侵入性30、31，是常规立体轴心颅内注射的理想替代品。此外，由于它目前在人类中使用30，32，使用这种技术的术前研究可以被认为是高度转化的。然而，由于缺乏可访问性，FUS BBB的开放尚未成为基础科学和学前研究中广泛确立的技术。因此，我们为 FUS BBB 开启的台面方法提供了详细的协议，作为有兴趣建立此技术的实验室的起点。
这些研究要么使用大功率空气支持的FUS特定超声波传感器，要么使用低功率阻尼聚焦超声波浸入式传感器进行。传感器由专为活性负载设计的 RF 功率放大器和标准台面功能发生器驱动。这些物品的详情可在 材料表中找到。
Protocol
所有实验程序均按照 UAB 机构动物护理和使用委员会 （IACUC） 准则进行。 1. 聚焦超声波驾驶设备设置 使用 50 Ohm 同轴 BNC 电缆将超声波传感器的输入连接到 RF 放大器的输出中，（2） RF 放大器对功能发生器输出的输入。 将函数生成器模式设置为每秒一次的鼻窦爆裂，其工作周期为 1%。 对于带 50 dB RF 放大器使用的 0.8 英寸焦距的阻尼 1 MHz 低功耗浸入式传?…
Representative Results
在这里，我们演示了用微泡聚焦超声波可以使用上述低功耗浸入传感器（图3）和FUS传感器（图4）指定的参数诱导本地化BBB开口。首先，在早期实验中，低功耗浸入式传感器被瞄准一个大脑半球（图3b）或中位（图3a）。动物在2小时后被牺牲与注入（图3a）或没有注入（?…
Discussion
在这里，我们描述了一个台面方法微泡协助FUS BBB打开与替代方法，包括，两个不同的传感器和方法的颅内瞄准有和没有MRI指导。目前，为了在实验室中建立 MRI 引导的 FUS BBB 开口，可以选择购买具有高度标准化和可重复效果的设备，并配备用户友好的界面。然而，许多实验室没有为此类仪器的成本做好准备。因此，此协议的主要目标是提供任何实验室都可以建立的起点，以建立他们在该技术方面?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究部分得到了NSF EPSCoR研究基础设施基金（1632881）的支持。此外，这项研究部分得到了西维坦国际研究中心的支持，伯明翰，AL。作者感激地感谢阿拉巴马大学伯明翰小型动物成像共享设施赠款 [NIH P30 CA013148] 的服务和设施的使用。作者感谢拉吉夫·乔普拉的支持和指导。
Materials
| Bubble shaker | Lantheus Medical Imaging | VMIX | VIALMIX, actiation device used to activate Definity microbubbles |
| Catheter plug/ Injection cap | SAI infusion technologies | Part Number: IC | Catheter plug/ Injection cap |
| Evans blue dye | Sigma | E2129-10G | Evans blue dye |
| Function generator | Tektronix | AFG3022B | Dual channel, 250MS/s, 25MHz |
| FUS transducer, 1.1MHz | FUS Instruments | TX-110 | 1 MHz MRI-compatible spherically focused ultrasound transducer with a hydrophone |
| Heating pad for Mice and Rats | Kent Scientific | PS-03 | Heating pad- PhysioSuite for Mice and Rats |
| Infusion pump | KD Scientific | 780100 | KDS 100 Legacy Single Syringe Infusion Pump |
| Kapton tape | Gizmo Dorks | https://www.amazon.com/dp/B01N1GGKRC/ ref=cm_sw_em_r_mt_dp_U_GbR7Db56HKD91 |
Gizmo Dorks Kapton Tape (Polyimide) for 3D Printers and Printing, 8 x 8 inches, 10 Sheets per Pack |
| Low power immersion transducer, 1MHz | Olympus | V303-SU | Immersion Transducer, 1 MHz, 0.50 in. Element Diameter, Standard Case Style, Straight UHF Connector, F=0.80IN PTF |
| Magnet sets | WINOMO | https://www.amazon.com/dp/B01DJZQJBG/ ref=cm_sw_em_r_mt_dp_U_JYQ7DbM32E5QC |
WINOMO 15mm Sew In Magnetic Bag Clasps for Sewing Scrapbooking – 10 Sets |
| RF amplifier | E&I | A075 | 75W |
| Tail vein catheter | BD | 382512/ Fisher Item: NC1228513 | 24g BD Insyte Autoguard shielded IV catheters (non-winged) |
| Ultrasound contrast microbubbles | Lantheus Medical Imaging | DE4, DE16 | DEFINITY (Perflutren Lipid Microsphere) |
| Ultrasound gel | Aquasonic | https://www.amazon.com/dp/B07FPQDM4F/ ref=cm_sw_em_r_mt_dp_U_D6Q7Db3J9QP7P |
Ultrasound Gel Aquasonic 100 Transmission 1 Liter Squeeze Bottle |
| Winged infusion sets, 22ga. | Fisher Healthcare | 22-258087 | Terumo Surflo Winged Infusion Sets |
| motor controller software | N/A | N/A | custom software written in LabView for controlling the Velmex motor controller |
| runtime environment for the motor controller software | National Instruments | LabView runtime engine version 2017 or better | https://www.ni.com/en-us/support/downloads/software-products/download.labview.html |
| 3 axis Linear stage actuator (XYZ positioner) | Velmex | ||
| bolts | Velmex | MB-1 | BiSlide Bolt 1/4-20×3/4" Socket cap screw (10 pack), Qty:3 |
| motor controller | Velmex | VXM-3 | Control,3 axis programmable stepping motor control, Qty:1 |
| mounting cleats | Velmex | MC-2 | Cleat, 2 hole BiSlide, Qty:6 |
| mounting cleats | Velmex | MC-2 | Cleat, 2 hole BiSlide, Qty:2 |
| usb to serial converter | Velmex | VXM-USB-RS232 | USB to RS232 Serial Communication Cable 10ft, Qty:1 |
| x-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| x-axis stepper motor | Velmex | PK266-03A-P1 | Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1 |
| y-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| y-axis stepper motor | Velmex | PK266-03A-P1 | Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1 |
| z-axis damper | Velmex | D6CL-6.3F | D6CL Damper for Type 23 Double Shaft Stepper Motor, Qty:1 |
| z-axis linear stage | Velmex | MN10-0100-M02-21 | BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1 |
| z-axis stepper motor | Velmex | PK266-03B-P2 | Vexta Type 23T2, Double Shaft Stepper Motor, Qty:1 |
| 3D printable files | |||
| Immersion transducer mount and pointer | https://www.tinkercad.com/things/cRgTthGXSRq | ||
| Stereotaxic frame | https://www.tinkercad.com/things/ilynoQcdqlH | ||
| Stereotaxic frame holder | https://www.tinkercad.com/things/aZNgqhBOHAX | ||
| 9.4T small bore animal MRI | Bruker | Bruker BioSpec 94/20 | ParaVision version 5.1 |
| AAV9-hsyn-GFP | Addgene | ||
| Cream hair remover | Church & Dwight | Nair cream | |
| gadobutrol MRI contrast agent | Bayer | Gadavist (Gadobutrol, 1mM/mL) | |
| Stereotactic frame | Stoelting | #51500 | not MRI compatible |
| turnkey FUS delivery device | FUS Instruments | RK-300 | ready to use MRI compatible FUS for rodents |
Riferimenti
- Markou, A., Chiamulera, C., Geyer, M. A., Tricklebank, M., Steckler, T. Removing obstacles in neuroscience drug discovery: the future path for animal models. Neuropsychopharmacology. 34 (1), 74-89 (2009).
- Schoepp, D. D. Where will new neuroscience therapies come from. Nature Reviews. Drug Discovery. 10 (10), 715-716 (2011).
- Insel, T. R., Landis, S. C. Twenty-five years of progress: the view from NIMH and NINDS. Neuron. 80 (3), 561-567 (2013).
- Bicker, J., Alves, G., Fortuna, A., Falcão, A. Blood-brain barrier models and their relevance for a successful development of CNS drug delivery systems: a review. European Journal of Pharmaceutics and Biopharmaceutics. 87 (3), 409-432 (2014).
- Pardridge, W. M. The blood-brain barrier: bottleneck in brain drug development. NeuroRx: the journal of the American Society for Experimental NeuroTherapeutics. 2 (1), 3-14 (2005).
- Millan, M. J., Goodwin, G. M., Meyer-Lindenberg, A., Ove Ögren, S. Learning from the past and looking to the future: Emerging perspectives for improving the treatment of psychiatric disorders. European Neuropsychopharmacology. 25 (5), 599-656 (2015).
- Correll, C. U., Carlson, H. E. Endocrine and metabolic adverse effects of psychotropic medications in children and adolescents. Journal of the American Academy of Child and Adolescent Psychiatry. 45 (7), 771-791 (2006).
- Girgis, R. R., Javitch, J. A., Lieberman, J. A. Antipsychotic drug mechanisms: links between therapeutic effects, metabolic side effects and the insulin signaling pathway. Molecular Psychiatry. 13 (10), 918-929 (2008).
- Patel, M. M., Goyal, B. R., Bhadada, S. V., Bhatt, J. S., Amin, A. F. Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs. 23 (1), 35-58 (2009).
- McCluskey, L., Campbell, S., Anthony, D., Allan, S. M. Inflammatory responses in the rat brain in response to different methods of intra-cerebral administration. Journal of Neuroimmunology. 194 (1-2), 27-33 (2008).
- Thanou, M., Gedroyc, W. MRI-Guided Focused Ultrasound as a New Method of Drug Delivery. Journal of drug delivery. 2013, 616197 (2013).
- Burgess, A., Hynynen, K. Noninvasive and targeted drug delivery to the brain using focused ultrasound. ACS Chemical Neuroscience. 4 (4), 519-526 (2013).
- Burgess, A., Shah, K., Hough, O., Hynynen, K. Focused ultrasound-mediated drug delivery through the blood-brain barrier. Expert Review of Neurotherapeutics. 15 (5), 477-491 (2015).
- Shin, J., et al. Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters. Neurosurgical Focus. 44 (2), 15 (2018).
- Bing, C., et al. Characterization of different bubble formulations for blood-brain barrier opening using a focused ultrasound system with acoustic feedback control. Scientific Reports. 8 (1), 7986 (2018).
- Hynynen, K., McDannold, N., Vykhodtseva, N., Jolesz, F. A. Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology. 220 (3), 640-646 (2001).
- Baseri, B., et al. Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood-brain barrier using focused ultrasound and microbubbles. Physics in Medicine and Biology. 57 (7), 65-81 (2012).
- Rodríguez-Frutos, B., et al. Enhanced brain-derived neurotrophic factor delivery by ultrasound and microbubbles promotes white matter repair after stroke. Biomaterials. 100, 41-52 (2016).
- Karakatsani, M. E., et al. Amelioration of the nigrostriatal pathway facilitated by ultrasound-mediated neurotrophic delivery in early Parkinson’s disease. Journal of Controlled Release. 303, 289-301 (2019).
- Lin, C. -. Y., et al. Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson’s disease mouse model. Journal of Controlled Release. 235, 72-81 (2016).
- Long, L., et al. Treatment of Parkinson’s disease in rats by Nrf2 transfection using MRI-guided focused ultrasound delivery of nanomicrobubbles. Biochemical and Biophysical Research Communications. , (2016).
- Fan, C. -. H., Lin, C. -. Y., Liu, H. -. L., Yeh, C. -. K. Ultrasound targeted CNS gene delivery for Parkinson’s disease treatment. Journal of Controlled Release. 261, 246-262 (2017).
- Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound. Biochemical and Biophysical Research Communications. 340 (4), 1085-1090 (2006).
- Todd, N., et al. Modulation of brain function by targeted delivery of GABA through the disrupted blood-brain barrier. Neuroimage. 189, 267-275 (2019).
- Nance, E., et al. Non-invasive delivery of stealth, brain-penetrating nanoparticles across the blood-brain barrier using MRI-guided focused ultrasound. Journal of Controlled Release. 189, 123-132 (2014).
- Mulik, R. S., et al. Localized delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat brain using focused ultrasound. Biomaterials. 83, 257-268 (2016).
- Lin, T., et al. Blood-Brain-Barrier-Penetrating Albumin Nanoparticles for Biomimetic Drug Delivery via Albumin-Binding Protein Pathways for Antiglioma Therapy. ACS Nano. 10 (11), 9999-10012 (2016).
- Timbie, K. F., et al. MR image-guided delivery of cisplatin-loaded brain-penetrating nanoparticles to invasive glioma with focused ultrasound. Journal of Controlled Release. 263, 120-131 (2017).
- Fan, C. -. H., et al. SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumor drug delivery. Biomaterials. 34 (14), 3706-3715 (2013).
- Mainprize, T., et al. Blood-Brain Barrier Opening in Primary Brain Tumors with Non-invasive MR-Guided Focused Ultrasound: A Clinical Safety and Feasibility Study. Scientific Reports. 9 (1), 321 (2019).
- Chen, K. -. T., Wei, K. -. C., Liu, H. -. L. Theranostic Strategy of Focused Ultrasound Induced Blood-Brain Barrier Opening for CNS Disease Treatment. Frontiers in Pharmacology. 10, 86 (2019).
- Lipsman, N., et al. Blood-brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nature Communications. 9 (1), 2336 (2018).
- . Compound Administration I Available from: https://www.jove.com/science-education/10198/compound-administration-i (2020)
- Liu, H. -. L., et al. Magnetic resonance imaging enhanced by superparamagnetic iron oxide particles: usefulness for distinguishing between focused ultrasound-induced blood-brain barrier disruption and brain hemorrhage. Journal of Magnetic Resonance Imaging. 29 (1), 31-38 (2009).
- Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. Survivable stereotaxic surgery in rodents. Journal of Visualized Experiments. (20), e880 (2008).
- Marty, B., et al. Dynamic study of blood-brain barrier closure after its disruption using ultrasound: a quantitative analysis. Journal of Cerebral Blood Flow and Metabolism. 32 (10), 1948-1958 (2012).
- Alonso, A., Reinz, E., Fatar, M., Hennerici, M. G., Meairs, S. Clearance of albumin following ultrasound-induced blood-brain barrier opening is mediated by glial but not neuronal cells. Brain Research. 1411, 9-16 (2011).
- McDannold, N., Zhang, Y., Vykhodtseva, N. Blood-brain barrier disruption and vascular damage induced by ultrasound bursts combined with microbubbles can be influenced by choice of anesthesia protocol. Ultrasound in Medicine & Biology. 37 (8), 1259-1270 (2011).
- McDannold, N., Zhang, Y., Vykhodtseva, N. The Effects of Oxygen on Ultrasound-Induced Blood-Brain Barrier Disruption in Mice. Ultrasound in Medicine & Biology. 43 (2), 469-475 (2017).
- O’Reilly, M. A., Muller, A., Hynynen, K. Ultrasound insertion loss of rat parietal bone appears to be proportional to animal mass at submegahertz frequencies. Ultrasound in Medicine & Biology. 37 (11), 1930-1937 (2011).
- Abrahao, A., et al. First-in-human trial of blood-brain barrier opening in amyotrophic lateral sclerosis using MR-guided focused ultrasound. Nature Communications. 10 (1), 4373 (2019).
Citazione di questo articolo
Rich, M., Whitsitt, Q., Lubin, F., Bolding, M. A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model. J. Vis. Exp. (160), e61113, doi:10.3791/61113 (2020).