Name: a benchtop approach to the location specific blood brain barrier opening using focused ultrasound in a rat model
Uploaded: 2020-06-13T12:30:00.000+00:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model at JoVE.com

この研究は、クレムソン大学(1632881)に対するNSF EPSCoR研究インフラ補助金によって部分的に支援されました。さらに、この研究の一部は、Civitan国際研究センター、バーミンガム、ALによってサポートされました。著者らは、アラバマ大学バーミンガム小動物イメージング共有施設グラント[NIH P30 CA013148]のサービスと施設の使用を感謝して認めている。著者らは、ラジーヴ・チョプラの支援と指導を認めている。

著者らは開示するものは何もない。

ここでは、MRIガイダンスの有無にかかわらず頭蓋内ターゲティングのための2つの異なるトランスデューサーおよび方法を含む代替アプローチで、マイクロバブル支援FUS BBB開口部へのベンチトップアプローチについて説明した。現在、MRIガイド付きFUS BBBのオープンをラボに設置するために、ユーザーフレンドリーなインターフェイスで高度に標準化された再現性の高い結果を提供する優れた?...

基本的な神経科学の多くの発見にもかかわらず、神経発達および神経変性疾患の新たな治療法の数は比較的限られた1,2のままである。神経疾患に関与する遺伝子、分子および細胞回路のより深い理解は、現在の技術を有するヒトでは実現不可能な有望な治療法を示唆している3。効果的な治療は、多くの場合、脳浸透性と部位特異的な4、5、6、7、8である必要性によって制限されます。しかしながら、特定の脳領域への局所的な薬物送達の既存の方法(例えば、注射またはカニューレによる送達)は侵襲的であり、頭蓋骨9に作られる開口部を必要とする。この手術の侵襲性は、人間の脳への局所的な送達の日常的な使用を防ぎます.さらに、組織損傷および結果として生じる炎症反応は、脳内注射10に依存する基礎的および前臨床試験のためのユビキタスな交論である。血液脳関門(BBB)を越えて非侵襲的に薬剤を送達し、特定の脳領域に標的化する能力は、神経疾患の治療に大きな影響を与えると同時に、前臨床研究のための強力な治験ツールを提供する可能性がある。
最小限の組織損傷を伴うBBBを介して標的輸送の1つの方法は、経頭蓋集結超音波(FUS)をマイクロバブルと共にBBB11、12、12、13、14、15、16を局所的かつ一時的に開く。FUS BBBの開口は神経栄養因子17、18、19、遺伝子治療20、21、22、抗体23、神経伝達物質24、およびナノ粒子25、26、27、28、29などの脳領域を標的とする治療を局所化することによって、神経変性疾患、脳卒中および神経膠腫の治療に関する最近の注目を集めている。用途の広い範囲とその非侵襲的な性質30、31、FUS BBBの開口部は、日常的な立体的な頭蓋内注射に理想的な代替手段です。さらに、ヒト30,32における現在の使用により、この技術を用いた前臨床調査は高度に翻訳的であると考えることができる。しかし、FUS BBBの開設は、アクセシビリティの欠如のために基礎科学と前臨床研究において広く確立された技術ではありません。そのため、この技術の確立に関心のあるラボの出発点として、FUS BBBのオープンに対するベンチトップアプローチの詳細なプロトコルを提供します。
これらの研究は、高出力空気を支持するFUS特異的超音波トランスデューサーまたは低電力減衰集積超音波浸漬トランスデューサーのいずれかで行った。トランスデューサは、反応性負荷用に設計されたRFパワーアンプと標準のベンチトップファンクションジェネレータによって駆動されました。これらの項目の詳細については、 資料一覧を参照してください。

<table><tbody><tr><td>Bubble shaker</td><td>Lantheus Medical Imaging</td><td>VMIX</td><td>VIALMIX, actiation device used to activate Definity microbubbles</td></tr><tr><td>Catheter plug/ Injection cap</td><td>SAI infusion technologies</td><td>Part Number: IC</td><td>Catheter plug/ Injection cap</td></tr><tr><td>Evans blue dye</td><td>Sigma</td><td>E2129-10G</td><td>Evans blue dye</td></tr><tr><td>Function generator</td><td>Tektronix</td><td>AFG3022B</td><td>Dual channel, 250MS/s, 25MHz</td></tr><tr><td>FUS transducer, 1.1MHz</td><td>FUS Instruments</td><td>TX-110</td><td>1 MHz MRI-compatible spherically focused ultrasound transducer with a hydrophone</td></tr><tr><td>Heating pad for Mice and Rats</td><td>Kent Scientific</td><td>PS-03</td><td>Heating pad- PhysioSuite for Mice and Rats</td></tr><tr><td>Infusion pump</td><td>KD Scientific</td><td>780100</td><td>KDS 100 Legacy Single Syringe Infusion Pump</td></tr><tr><td>Kapton tape</td><td>Gizmo Dorks</td><td>https://www.amazon.com/dp/B01N1GGKRC/&lt;br/&gt; ref=cm_sw_em_r_mt_dp_U_GbR7Db56HKD91</td><td>Gizmo Dorks Kapton Tape (Polyimide) for 3D Printers and Printing, 8 x 8 inches, 10 Sheets per Pack</td></tr><tr><td>Low power immersion transducer, 1MHz</td><td>Olympus</td><td>V303-SU</td><td>Immersion Transducer, 1 MHz, 0.50 in. Element Diameter, Standard Case Style, Straight UHF Connector, F=0.80IN PTF</td></tr><tr><td>Magnet sets</td><td>WINOMO</td><td>https://www.amazon.com/dp/B01DJZQJBG/&lt;br/&gt; ref=cm_sw_em_r_mt_dp_U_JYQ7DbM32E5QC</td><td>WINOMO 15mm Sew In Magnetic Bag Clasps for Sewing Scrapbooking - 10 Sets</td></tr><tr><td>RF amplifier</td><td>E&amp;amp;I</td><td>A075</td><td>75W</td></tr><tr><td>Tail vein catheter</td><td>BD</td><td>382512/ Fisher Item: NC1228513</td><td>24g BD Insyte Autoguard shielded IV catheters (non-winged)</td></tr><tr><td>Ultrasound contrast microbubbles</td><td>Lantheus Medical Imaging</td><td>DE4, DE16</td><td>DEFINITY (Perflutren Lipid Microsphere)</td></tr><tr><td>Ultrasound gel</td><td>Aquasonic</td><td>https://www.amazon.com/dp/B07FPQDM4F/&lt;br/&gt; ref=cm_sw_em_r_mt_dp_U_D6Q7Db3J9QP7P</td><td>Ultrasound Gel Aquasonic 100 Transmission 1 Liter Squeeze Bottle</td></tr><tr><td>Winged infusion sets, 22ga.</td><td>Fisher Healthcare</td><td>22-258087</td><td>Terumo Surflo Winged Infusion Sets</td></tr><tr><td>motor controller software</td><td>N/A</td><td>N/A</td><td>custom software written in LabView for controlling the Velmex motor controller</td></tr><tr><td>runtime environment for the motor controller software</td><td>National Instruments</td><td>LabView runtime engine version 2017 or better</td><td>https://www.ni.com/en-us/support/downloads/software-products/download.labview.html</td></tr><tr><td>3 axis Linear stage actuator (XYZ positioner)</td><td>Velmex</td><td></td><td></td></tr><tr><td>bolts</td><td>Velmex</td><td>MB-1</td><td>BiSlide Bolt 1/4-20x3/4&quot; Socket cap screw (10 pack), Qty:3</td></tr><tr><td>motor controller</td><td>Velmex</td><td>VXM-3</td><td>Control,3 axis programmable stepping motor control, Qty:1</td></tr><tr><td>mounting cleats</td><td>Velmex</td><td>MC-2</td><td>Cleat, 2 hole BiSlide, Qty:6</td></tr><tr><td>mounting cleats</td><td>Velmex</td><td>MC-2</td><td>Cleat, 2 hole BiSlide, Qty:2</td></tr><tr><td>usb to serial converter</td><td>Velmex</td><td>VXM-USB-RS232</td><td>USB to RS232 Serial Communication Cable 10ft, Qty:1</td></tr><tr><td>x-axis linear stage</td><td>Velmex</td><td>MN10-0100-M02-21</td><td>BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td></tr><tr><td>x-axis stepper motor</td><td>Velmex</td><td>PK266-03A-P1</td><td>Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1</td></tr><tr><td>y-axis linear stage</td><td>Velmex</td><td>MN10-0100-M02-21</td><td>BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td></tr><tr><td>y-axis stepper motor</td><td>Velmex</td><td>PK266-03A-P1</td><td>Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1</td></tr><tr><td>z-axis damper</td><td>Velmex</td><td>D6CL-6.3F</td><td>D6CL Damper for Type 23 Double Shaft Stepper Motor, Qty:1</td></tr><tr><td>z-axis linear stage</td><td>Velmex</td><td>MN10-0100-M02-21</td><td>BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1</td></tr><tr><td>z-axis stepper motor</td><td>Velmex</td><td>PK266-03B-P2</td><td>Vexta Type 23T2, Double Shaft Stepper Motor, Qty:1</td></tr><tr><td>3D printable files</td><td></td><td></td><td></td></tr><tr><td>Immersion transducer mount and pointer</td><td></td><td></td><td>https://www.tinkercad.com/things/cRgTthGXSRq</td></tr><tr><td>Stereotaxic frame</td><td></td><td></td><td>https://www.tinkercad.com/things/ilynoQcdqlH</td></tr><tr><td>Stereotaxic frame holder</td><td></td><td></td><td>https://www.tinkercad.com/things/aZNgqhBOHAX</td></tr><tr><td>9.4T small bore animal MRI</td><td>Bruker</td><td>Bruker BioSpec 94/20</td><td>ParaVision version 5.1</td></tr><tr><td>AAV9-hsyn-GFP</td><td>Addgene</td><td></td><td></td></tr><tr><td>Cream hair remover</td><td>Church &amp;amp; Dwight</td><td></td><td>Nair cream</td></tr><tr><td>gadobutrol MRI contrast agent</td><td>Bayer</td><td></td><td>Gadavist (Gadobutrol, 1mM/mL)</td></tr><tr><td>Stereotactic frame</td><td>Stoelting</td><td>#51500</td><td>not MRI compatible</td></tr><tr><td>turnkey FUS delivery device</td><td>FUS Instruments</td><td>RK-300</td><td>ready to use MRI compatible FUS for rodents</td></tr></tbody></table>

a benchtop approach to the location specific blood brain barrier opening using focused ultrasound in a rat model

立体外科は、げっ歯類の脳への局在化した薬物および遺伝子送達のためのゴールドスタンダードである。この技術は、ターゲット脳領域への正確な局在化およびオフターゲットの副作用の減少を含む全身配信よりも多くの利点を有する。しかし、立体外科は非常に侵襲的であり、その翻訳効果を制限し、長い回復時間を必要とし、複数の脳領域を標的とする場合に課題を提供する。集中超音波(FUS)は、一過性のミリサイズ領域の血液脳関門(BBB)を開くために循環マイクロバブルと組み合わせて使用することができます。これにより、通常は BBB を横断できない全身配信エージェントの頭蓋内局在化が可能になります。この技術は、立体外科に対する非侵襲的な代替手段を提供する。しかし、これまで、この技術は、機器へのアクセスが限られているため、また標準化された方法により、神経科学の研究室ではまだ広く採用されていません。このプロトコルの全体的な目標は、手頃な価格で再現可能なFUS BBB開口部(BBBO)へのベンチトップアプローチを提供するため、任意のラボで簡単に採用することができます。

ここでは、マイクロバブルを用いた集結超音波が、低電力浸漬トランスデューサー(図3)とFUSトランスデューサ(図4)の両方で上記で指定されたパラメータを使用して局所的なBBB開口部を誘導できることを示す。まず、初期の実験において、低電力浸漬変換器を、前部(図3b)または内側(図3a)のいずれか1つの?...

Watch this Scientific Journal Video about A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model at JoVE.com

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

マイクロバブル剤を用いた集中超音波は、血液脳関門を局所的かつ一過性に開くことができる。この技術は、血液脳関門を越えて幅広い薬剤を送達するために使用されてきた。この記事では、MRI ガイダンスの有無にかかわらずげっ歯類の脳へのローカライズされた配信のための詳細なプロトコルを提供します。

ラットモデルにおける集中超音波を用いた位置特異的血液脳関門開口へのベンチトップアプローチ

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

a-benchtop-approach-to-location-specific-blood-brain-barrier-opening

Research

JoVE Journal

Neuroscience

4.5K ビュー.  University of Alabama at Birmingham. マイクロバブル剤を用いた集中超音波は、血液脳関門を局所的かつ一過性に開くことができる。この技術は、血液脳関門を越えて幅広い薬剤を送達するために使用されてきた。この記事では、MRI ガイダンスの有無にかかわらずげっ歯類の脳へのローカライズされた配信のための詳細なプロトコルを提供します。

ビデオ: A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

ラットモデルでの経頭蓋集束超音波を用いた血液脳関門のMRI誘導破壊

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

医学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

神経科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

大人のラットの特定の脳領域の興奮毒性病変に対する定位手術

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

虚血性脳卒中のモデルラットの血液脳関門の破壊のIn Vivo評価

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise neuromodulatory technology. Although LIFU-mediated blood-brain barrier (BBB) opening has been explored in detail, no standardized technique for blood-spinal cord barrier (BSCB) opening has been established to date. Therefore, this protocol presents a method for successful BSCB disruption using LIFU sonication in a rat model, including descriptions of animal preparation, microbubble administration, target selection&#160;and localization, as well as BSCB disruption visualization and confirmation. The approach reported here is particularly useful for researchers who need a fast and cost-effective method to test and confirm target localization and precise BSCB disruption in a small animal model with a focused ultrasound transducer, evaluate the BSCB efficacy of sonication parameters, or explore applications for LIFU at the spinal cord, such as drug delivery, immunomodulation, and neuromodulation. Optimizing this protocol for individual use is recommended, especially for advancing future preclinical, clinical, and translational work.

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

ラットモデルにおける低強度集束超音波を用いた脊髄バリアの破壊

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...

生物工学

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

げっ歯類における血液脳関門開口のためのハイスループット画像誘導定型神経ナビゲーションと集中超音波システム

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

脳構造を標的とし、化学遺伝学的神経調節を評価するための集束超音波誘発血液脳関門開口部

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Enhancing Drug Delivery to the Brain: MRI-Guided Ultrasound Techniques in Rats

Monitoring Blood-Brain Barrier Opening in Rats with a Preclinical Focused Ultrasound System

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic macromolecular agents to the brain. The integration of MR-guided low-intensity pulsed focused ultrasound (FUS) with microbubble pre-injection is a promising technique for non-invasive and non-toxic BBB modulation. MRI can offer superior soft-tissue contrast and various quantitative assessments, such as vascular permeability, perfusion, and the spatial-temporal distribution of MRI contrast agents. Notably, contrast-enhanced MRI techniques with gadolinium-based MR contrast agents have been shown to be the gold standard for detecting BBB openings. This study outlines a comprehensive methodology involving MRI protocols and animal procedures for monitoring BBB opening in a rat model. The rat model provides the added benefit of jugular vein catheter utilization, which facilitates rapid medication administration. A stereotactic-guided preclinical FUS transducer facilitates the refinement and streamlining of animal procedures and MRI protocols. The resulting methods are characterized by reproducibility and simplicity, eliminating the need for specialized surgical expertise. This research endeavors to contribute to the optimization of preclinical procedures with rat models and encourage further investigation into the modulation of the BBB to enhance therapeutic interventions in neurological disorders.

This study describes a combined magnetic resonance imaging (MRI) and low-intensity pulsed focused ultrasound (FUS) protocol, utilizing living rats with jugular vein catheterization to monitor blood-brain barrier (BBB) opening.

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic ...

Focus Ultrasound Based Microbubble Mediated Blood-Brain Barrier Opening: A Technique to Create Localized Transient Openings in Blood-Brain Barrier of Mouse by Sonoporation

Source: Haumann, R. et al. <a href="https://www.jove.com/video/61269" target="_blank">A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents.</a> J. Vis. Exp. (2020)
This video describes a method of temporarily disrupting the blood-brain barrier (BBB) by creating transient openings using microbubble-mediated focused ultrasound (FUS). These transient local BBB openings can eventually be used to deliver larger-sized drug molecules directly into the brain parenchyma.

フォーカス超音波を用いたマイクロバブル媒介血液脳関門開口:超音波処理によりマウスの血液脳関門に局在性一過性開口を作製する技術

Source: Haumann, R. et al. A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in ...