Name: a high-throughput image-guided stereotactic neuronavigation and focused ultrasound system for blood-brain barrier opening in rodents
Uploaded: 2020-07-16T12:30:00
Description: Watch this Scientific Journal Video about A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents at JoVE.com

This project was funded by the KWF-STW (Drug Delivery by Sonoporation in Childhood Diffuse Intrinsic Pontine Glioma and High-grade Glioma). We thank Ilya Skachkov and Charles Mougenot for their input in the development of the system.

All in vivo experiments were approved by the Dutch ethical committee (license permit number AVD114002017841) and the Animal Welfare Body of the Vrije Universiteit Amsterdam, the Netherlands. The investigators were trained in the basics of the FUS system in order to minimize the discomfort of the animals.
1. Focused ultrasound system
NOTE: The described setup is an inhouse built BBB disruption system based on commercially available components and includes a 3D-printed ...

In this study, we developed a cost-effective image guided based FUS system for transient BBB disruption for increased drug delivery into the brain parenchyma. The system was largely built with commercially available components and in conjunction with X-ray and BLI. The modularity of the proposed design allows the use of several imaging modalities for planning and assessment in high-throughput workflows. The system can be combined with more comprehensive high-resolution 3D imaging modalities, for example high-resolution M...

The blood-brain barrier (BBB) is a major obstacle for drug delivery into the brain parenchyma. Most therapeutic drugs that have been developed do not cross the BBB due to their physicochemical parameters (e.g., lipophilicity, molecular weight, hydrogen bond acceptors and donors) or are not retained due to their affinity for efflux transporters in the brain1,2. The small group of drugs that can cross the BBB are typically small lipophilic molecules, which are only effective in a limited number of brain diseases1,2. As a consequence, for the majority of brain diseases, pharmacological treatment options are limited and new drug delivery strategies are needed3,4.
Therapeutic ultrasound is an emerging technique that can be used for different neurological applications such as BBB disruption (BBBD), neuromodulation, and ablation4,5,6,7. In order to achieve a BBB opening with an extracorporeal ultrasound emitter through the cranium, focused ultrasound (FUS) is combined with microbubbles. Microbubble-mediated FUS results in increased bioavailability of drugs in the brain parenchyma5,8,9. In the presence of sound waves, microbubbles start to oscillate initiating transcytosis and disruption of the tight junctions between the endothelial cells of the BBB, enabling paracellular transport of larger molecules10. Previous studies confirmed the correlation between the intensity of the acoustic emission and the biological impact on the BBB opening11,12,13,14. FUS in combination with microbubbles has already been used in clinical trials for the treatment of glioblastoma using temozolomide or liposomal doxorubicin as the chemotherapeutic agent, or for therapy of Alzheimer&#8217;s disease and amyotrophic lateral sclerosis5,9,15,16.
Since ultrasound mediated BBB opening results in entirely new possibilities for pharmacotherapy, preclinical research for clinical translation is needed to assess the therapy response of selected drug candidates. This typically requires a high-throughput workflow with both high-spatial precision and preferably an integrated cavitation detection for monitoring of targeted BBB opening with a high reproducibility. If possible, these systems need to be cost effective in both initial investment and running costs in order to be scalable according to the study size. Most preclinical FUS systems are combined with MRI for image-guidance and treatment planning15,17,18,19. Although MRI gives detailed information about the tumor anatomy and volume, it is an expensive technique, which is generally performed by trained/skilled operators. In addition, high-resolution MRI may not always be available for researchers in preclinical facilities and requires long scanning times per animal, making it less suitable for high-throughput pharmacological studies. Noteworthy is that, for preclinical research in the field of neuro-oncology, in particular infiltrative tumor models, the possibility to visualize and target the tumor is essential for treatment success20. Currently, this requirement is only fulfilled by MRI or by tumors transduced with a photoprotein, enabling visualization with bioluminescence imaging (BLI) in combination with administration of the photoprotein substrate.
MRI-guided FUS systems often use a water bath to ensure ultrasound wave propagation for transcranial applications, whereby the head of the animal is partly submerged in the water, the so called &#8216;&#8217;bottom-up&#8217;&#8217; systems15,17,18. While these designs work generally well in smaller animal studies, they are a compromise between animal preparation times, portability and realistically maintainable hygienic standards during usage. As an alternative to MRI, other guidance methods for stereotactic navigation encompass the use of a rodent anatomical atlas21,22,23, laser pointer assisted visual sighting24, pinhole-assisted mechanical scanning device25, or BLI26. Most of these designs are &#8220;top-down&#8221; systems in which the transducer is placed on top of the animal&#8217;s head, with the animal in a natural position. The &#8216;&#8217;top-down&#8217;&#8217; workflow consists either of a water bath22,25,26 or a water-filled cone21,24. The benefit of using a transducer inside a closed cone is the more compact footprint, shorter setup time and straight-forward decontamination possibilities simplifying the entire workflow.
The interaction of the acoustic field with the microbubbles is pressure dependent and ranges from low-amplitude oscillations (referred to as stable cavitation) to transient bubble collapse (referred to as inertial cavitation)27,28. There is an established consensus that ultrasound-BBBD requires an acoustic pressure well above the stable cavitation threshold to achieve successful BBBD, but below the inertial cavitation threshold, which is generally associated with vascular/neuronal damage29. The most common form of monitoring and control is the analysis of the (back-)scattered acoustic signal using passive cavitation detection (PCD), as suggested by McDannold et al.12. PCD relies on the analysis of the Fourier spectra of microbubble emission signals, in which the strength and appearance of stable cavitation hallmarks (harmonics, subharmonics, and ultraharmonics) and inertial cavitation markers (broadband response) can be measured in real-time.
A &#8220;one size fits all&#8221; PCD-analysis for precise pressure control is complicated due to the polydispersity of the microbubble formulation (the oscillation amplitude depends strongly on the bubble diameter), the differences in bubble shell properties between brands, and the acoustic oscillation, which depends strongly on frequency and pressure30,31,32. As a consequence, many different PCD detection protocols have been suggested, which have been adapted to particular combinations of all these parameters and have been used in various application scenarios (ranging from in vitro experimentation over small animal protocols to PCD for clinical usage) for robust cavitation detection and even for retroactive feedback control of the pressure11,14,30,31,32,33,34,35. The PCD protocol employed in the scope of this study is derived directly from McDannold et al.12 and monitors the harmonic emission for the presence of stable cavitation and broadband noise for inertial cavitation detection.
We have developed an image-guided neuronavigation FUS system for transient opening of the BBB to increase drug delivery into the brain parenchyma. The system is based on commercially available components and can be easily adapted to several different imaging modalities, depending on the available imaging techniques in the animal facility. Since we require a high-throughput workflow, we have opted to use X-ray and BLI for image-guidance and treatment planning. Tumor cells transduced with a photoprotein (e.g., luciferase) are suitable for BLI imaging20. After administration of the photoprotein substrate, tumor cells can be monitored in vivo and tumor growth and location can be determined20,36. BLI is a low-cost imaging modality, it enables to follow the tumor growth over time, it has fast scanning times and it correlates well with tumor growth measured with MRI36,37. We have opted to replace the water bath with a water-filled cone attached to the transducer to enable flexibility to freely move the platform on which the rodent is mounted8,24. The design is based on a detachable platform equipped with integration of (I) small-animal stereotactic platform (II) fiducial markers with both X-ray and optical-image compatibility (III) rapid-detachable anesthesia mask, and (IV) integrated temperature regulated animal heating system. After the initial induction of anesthesia, the animal is mounted in a precise position on the platform where it remains during the entire procedure. Consequently, the entire platform passes all stations of the workflow of the entire intervention, while maintaining an accurate and reproducible positioning and sustained anesthesia. The control software allows the automatic detection of the fiducial markers and automatically registers all types of images and image modalities (i.e., micro-CT, X-ray, BLI and fluorescence imaging) into the frame of reference of the stereotactic platform. With help of an automatic calibration procedure, the focal length of the ultrasound transducer is precisely known within, which enables the automatic fusion of interventional planning, acoustic delivery and follow-up imaging analysis. As shown in Figure 1 and Figure 2, this setup provides a high degree of flexibility to design dedicated experimental workflows and allows interleaved handling of the animal at different stations, which in-turn facilitates high-throughput experiments. We have used this technique for successful drug delivery in mouse xenografts of high-grade glioma such as diffuse midline glioma.

<table>
	<tbody>
		<tr>
			<td>1 mL luer-lock syringe</td>
			<td>Becton Dickinson</td>
			<td>309628</td>
			<td>Plastipak</td>
		</tr>
		<tr>
			<td>19 G needle</td>
			<td>Terumo Agani</td>
			<td>8AN1938R1</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>Terumo Agani</td>
			<td>8AN2316R1</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Transpore surgical tape</td>
			<td>Science applied to life</td>
			<td>7000032707</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Arbitrary waveform generator</td>
			<td>Siglent</td>
			<td>n.a.</td>
			<td>SDG1025, 25 MHz, 125 Msa/s</td>
		</tr>
		<tr>
			<td>Automated stereotact</td>
			<td>in-house built</td>
			<td>n.a.</td>
			<td>Stereotact with all elements were in-house built</td>
		</tr>
		<tr>
			<td>Bruker In-Vivo Xtreme</td>
			<td>Bruker</td>
			<td>n.a.</td>
			<td>Includes software</td>
		</tr>
		<tr>
			<td>Buffered NaCl solution</td>
			<td>B. Braun Melsungen AG</td>
			<td>220/12257974/110</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorfine hydrochloride</td>
			<td>Indivior UK limitd</td>
			<td>n.a.</td>
			<td>0.324 mg</td>
		</tr>
		<tr>
			<td>Cage enrichment: paper-pulp smart home</td>
			<td>Bio services</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon filter</td>
			<td>Bickford</td>
			<td>NC0111395</td>
			<td>Omnicon f/air</td>
		</tr>
		<tr>
			<td>Ceramic spoon</td>
			<td>n.a</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin, potassium salt</td>
			<td>Gold Biotechnology</td>
			<td>LUCK-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VUmc pharmacy</td>
			<td>n.a.</td>
			<td>70%</td>
		</tr>
		<tr>
			<td>Evans Blue</td>
			<td>Sigma Aldrich</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresenius NaCl 0.9%</td>
			<td>Fresenius Kabi</td>
			<td>n.a.</td>
			<td>NaCl 0.9 %, 1000 mL</td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>Braun Surgical</td>
			<td>n.a.</td>
			<td>Histoacryl 0.5 mL</td>
		</tr>
		<tr>
			<td>Hydrophone</td>
			<td>Precision Acoustics</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton Dickinson</td>
			<td>324825/324826</td>
			<td>0.5 mL and 0.3 mL</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>TEVA Pharmachemie BV</td>
			<td>8711218013196</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Alfasan</td>
			<td>n.a.</td>
			<td>10 %, 10 mL</td>
		</tr>
		<tr>
			<td>Mouse food: Teklad global 18% protein rodent diet</td>
			<td>Envigo</td>
			<td>2918-11416M</td>
			<td></td>
		</tr>
		<tr>
			<td>Neoflon catheter</td>
			<td>Becton Dickinson</td>
			<td>391349</td>
			<td>26 GA 0.6 x 19 mm</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Keysight technologies</td>
			<td>n.a.</td>
			<td>InfiniiVision DSOX024A</td>
		</tr>
		<tr>
			<td>Plastic tubes</td>
			<td>Greiner bio-one</td>
			<td>210261</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Power amplifier</td>
			<td>Electronics &#38; Innovation Ltd</td>
			<td>210L</td>
			<td>Model 210L</td>
		</tr>
		<tr>
			<td>Preamplifier DC Coupler</td>
			<td>Precision Acoustics</td>
			<td>n..</td>
			<td>Serial number: DCPS94</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Sigma Aldrich</td>
			<td>S3146-1EA</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Sedazine</td>
			<td>AST Farma</td>
			<td>n.a.</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>SonoVue microbubbles</td>
			<td>Bracco</td>
			<td>n.a.</td>
			<td>8 &#181;l/ml</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Fresenius Kabi</td>
			<td>n.a.</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>various syringes can be used</td>
		</tr>
		<tr>
			<td>Temgesic</td>
			<td>Indivior UK limitd</td>
			<td>n.a.</td>
			<td>0.3 mg/ml</td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>Precision Acoustics</td>
			<td>n.a.</td>
			<td>1 MHz</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma Aldrich</td>
			<td>F4142-1EA</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>01-02</td>
			<td>Aquasonic 100</td>
		</tr>
		<tr>
			<td>Vidisic gel</td>
			<td>Bausch + Lomb</td>
			<td>n.a.</td>
			<td>10 g</td>
		</tr>
	</tbody>
</table>

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 described FUS system (Figure 1 and Figure 2) and the associated workflow have been used in over a 100 animals and produced reproducible data on both healthy and tumor bearing mice. Based on the recorded cavitation and the spectral density at the harmonics at the peak moment of the microbubble bolus injection, the spectral power of each frequency can be calculated using the Fourier analysis as explained in step 4 of the Protocol. Based on the acoustic protoco...

Watch this Scientific Journal Video about A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents at JoVE.com

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

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.

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 ...

Research

JoVE Journal

Neuroscience

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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

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 ...

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

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

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

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 ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

Applications of TEER Detection to Assess Cell Barrier Integrity (Video) | JoVE

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model (Video) | JoVE

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...