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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 (>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.

Introduction

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’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 ‘’bottom-up’’ 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 “top-down” systems in which the transducer is placed on top of the animal’s head, with the animal in a natural position. The ‘’top-down’’ 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 “one size fits all” 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.

Protocol

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 custom-made cone and detachable stereotactic platform. The system is designed modular, which facilitates modifications according to available equipment and specific use. The protocol describes the procedure for the sonoporation of a larger area in the pontine region of the mouse brain. By adjusting the target location, different parts of the brain could be targeted. In this study a 1 MHz mono-element transducer with a focal length of 75 mm, an aperture of 60 mm and a focal area of 1.5 x 1.5 x 5 mm (FWHM of peak pressure) was used. The focal plane of the transducer is positioned through the cranium of the animal in the horizontal plane intersecting with the ear bars.

  1. Select an appropriate transducer for BBB opening in rodents.
    NOTE: Based on the properties of the microbubbles and the employed frequency, the acoustic settings, in particular the mechanical index (MI), are subject to change13,38.
  2. Place the transducer in the 3D-printed cone.
  3. Employ an acoustically transparent mylar membrane at the bottom-end of the cone to achieve acoustic coupling of the beam propagation path, and fill the cone with degassed water.
  4. Mount the transducer above the animal on a motorized linear stage as shown in Figure 1 allowing automatic vertical positioning of the transducer.
  5. Design a detachable stereotactic platform based on the requirements of the study, which includes temperature regulated heating, bite and ear bars, anesthesia and multi-modality fiducial markers, as shown in Figure 1 and Figure 2. The mounting of the stereotactic platform consists of a 2D linear stage system, which allows precise automatic positioning (< 0.1 mm) of the animal under the beam.
  6. Connect the transducer to the acoustic emission chain shown in Figure 1 consisting of a transducer, a function generator and a power amplifier.
  7. Devise an image-processing pipeline to detect the multi-modality fiducial markers that allows precise sonoporation targeting of the brain area of interest and collection of the cavitation data detected by the needle hydrophone.
  8. Calibrate the system and determine the focus point of the transducer in correspondence to vertical positioning of the animal on the stereotactic platform.

2. Animal preparation

NOTE: The following protocol is specified for mice but can be adapted for rats. For these experiments female athymic nude Foxn1-/- mice (6-8 week old) were used.

  1. Allow the animal to acclimatize for at least one week in the animal facility and weigh the animal regularly.
  2. Administer buprenorphine (0.05 mg/kg) via subcutaneous (s.c.) injection 30 min prior to FUS treatment to start analgesic treatment.
  3. Anesthetize the animal with 3% isoflurane, 2 L/min O2 and verify that the animal is deeply anesthetized. Keep the animals anesthetized during the whole procedure and monitor the breathing frequency and heart rate to adjust the concentration of isoflurane as required.
  4. Apply eye ointment to prevent dry eyes and avoid possible injury.
  5. Remove hair on the top of the head with a razor and depilatory cream and wash afterwards with water to remove any residues to avoid irritation to the skin.
  6. For experiments with BLI tumor models, inject 150 µL of D-luciferin (30 mg/mL) intraperitoneal (i.p.) with a 29 G insulin syringe for BLI image-guidance.
  7. Insert a 26-30 G tail vein catheter and flush the catheter and vein with a small volume of heparin solution (5 UI/mL). Fill the catheter with heparin solution to avoid blood clotting.
    NOTE: Good catheterization is seen when there is a reflux of blood into the catheter. Avoid air bubbles in the catheter to prevent emboli. To avoid excessive injection pressure, make sure the length of the catheter is as short as possible.
  8. Place the animal on the temperature regulated stereotactic platform to avoid hypothermia.
    NOTE: Hypothermia reduces blood circulation, which can affect the injection/circulation of microbubbles and the pharmacokinetics of the drugs39.
  9. Immobilize and fix the head of the animal on the stereotactic platform using ear bars and a bite bar. Fixate the body with a strap and tape the tail of the animal to the platform.

3. In vivo image-guided focused ultrasound

NOTE: For this protocol a 1 MHz mono-element transducer with a tone-burst pulse with a 10 ms duration, a MI of 0.4 and a pulse repetition frequency of 1.6 Hz with 40 cycles for 240 s was used. The protocol is optimized for microbubbles stabilized by phospholipids containing sulphur hexafluoride (SF6) as an innocuous gas, whereby the mean bubble diameter is 2.5 μm and more than 90% of the bubbles are smaller than 8 μm.

  1. Place the stereotactic platform with the mounted animal in the imaging modality (e.g., BLI or X-ray) and take image(s) of the animal.
  2. Use the multi-modality fiducial markers in combination with the image-processing pipeline to mark the position of the animal according to the focus point of the transducer.
  3. Determine the target area by placing a brain outline over the acquired X-ray image or using BLI images to determine the center of the tumor (Figure 2). The position of specific parts of the brain are specified in the Paxinos Brain Atlas40 using the skull markings bregma and lambda as reference points. For example the pons is located x=-1.0, y=-0.8 and z=-4.5 from lambda.
  4. Shield the animal’s nostrils and mouth with adhesive tape to prevent ultrasound gel interfering with breathing.
  5. Apply ultrasound gel on top of the animal’s head.
  6. Retract the skin of the animals’ neck, lubricate the needle hydrophone with ultrasound gel and place the needle hydrophone in the direct vicinity of the occipital bone.
  7. Guide the transducer to the correct position using the image-processing pipeline and the focus point.
  8. Apply the preconfigured settings to all attached devices and target the brain region of interest.
    NOTE: Depending on the research question, tumor or brain regions can be sonoporated as a single focal point or as volumetric shape, as shown in Figure 2.
  9. Activate microbubbles as described by the manufacturer. Inject one bolus of 120 µL (5.4 µg) of microbubbles.
  10. Flush the tail vein catheter with saline to check the opening of the catheter.
  11. Inject the microbubbles and start the insonation.
  12. Record microbubble cavitation with the needle hydrophone.
  13. Administer an intravascular contrast agent or drug after sonoporation. The dose, timing and planning are dependent on the purpose of the study and the drug.
    NOTE: Evans blue is a common color agent to assess BBB opening41.
  14. Monitor the animal until the predetermined time point or before the humane endpoint.

4. Analysis of microbubble cavitation

NOTE: Here the applied procedure is described, which is suitable for in vivo experimentation for SF6-phospholipid microbubbles with an average diameter of 2.5 µm (80% of the bubbles below 8 µm) excited with a burst-tone pulse of 10 ms duration at a frequency of 1 MHz, as originally suggested by McDannold et al.12.

  1. Fourier-transform the recorded PCD signal from the time-domain into the frequency domain.
  2. Integrate the resulting spectral power for stable cavitation detection around the 2nd and 3rd harmonic (± 50 kHz), as shown in Figure 3 (green box at 2 and 3 MHz).
  3. Integrate the spectral power for inertial cavitation detection, between principal frequency, the 2nd, 3rd harmonic, the 1st and 2nd ultraharmonic and the first sub-harmonic (± 150 kHz), as shown in Figure 3 (red boxes).
  4. Integrate the spectral power around the principle frequency (1 MHz ± 50 kHz) for the normalization of both previously obtained PCD signals.
    NOTE: The PCD signal, for SF6-phospholipid microbubbles in vivo experiments at 1 MHz, does not display ultraharmonics or subharmonics before inertial cavitation sets in, as shown in Figure 3.

Results

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...

Discussion

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
1 mL luer-lock syringeBecton Dickinson309628Plastipak
19 G needleTerumo Agani8AN1938R1
23 G needleTerumo Agani8AN2316R1
3M Transpore surgical tapeScience applied to life7000032707or similar
Arbitrary waveform generatorSiglentn.a.SDG1025, 25 MHz, 125 Msa/s
Automated stereotactin-house builtn.a.Stereotact with all elements were in-house built
Bruker In-Vivo XtremeBrukern.a.Includes software
Buffered NaCl solutionB. Braun Melsungen AG220/12257974/110
Buprenorfine hydrochlorideIndivior UK limitdn.a.0.324 mg
Cage enrichment: paper-pulp smart homeBio servicesn.a.
Carbon filterBickfordNC0111395Omnicon f/air
Ceramic spoonn.an.a.
Cotton swabsn.a.n.a.
D-luciferin, potassium saltGold BiotechnologyLUCK-1
EthanolVUmc pharmacyn.a.70%
Evans BlueSigma AldrichE2129
Fresenius NaCl 0.9%Fresenius Kabin.a.NaCl 0.9 %, 1000 mL
HistoacrylBraun Surgicaln.a.Histoacryl 0.5 mL
HydrophonePrecision Acousticsn.a.
Insulin syringeBecton Dickinson324825/3248260.5 mL and 0.3 mL
IsofluraneTEVA Pharmachemie BV8711218013196250 mL
KetamineAlfasann.a.10 %, 10 mL
Mouse food: Teklad global 18% protein rodent dietEnvigo2918-11416M
Neoflon catheterBecton Dickinson39134926 GA 0.6 x 19 mm
OscilloscopeKeysight technologiesn.a.InfiniiVision DSOX024A
Plastic tubesGreiner bio-one21026150 mL
Power amplifierElectronics & Innovation Ltd210LModel 210L
Preamplifier DC CouplerPrecision Acousticsn..Serial number: DCPS94
ScissorsSigma AldrichS3146-1EAor similar
SedazineAST Farman.a.2%
SonoVue microbubblesBraccon.a.8 µl/ml
Sterile waterFresenius Kabin.a.1000 mL
Syringen.a.n.a.various syringes can be used
TemgesicIndivior UK limitdn.a.0.3 mg/ml
TransducerPrecision Acousticsn.a.1 MHz
TweezersSigma AldrichF4142-1EAor similar
Ultrasound gelParker Laboratories Inc.01-02Aquasonic 100
Vidisic gelBausch + Lombn.a.10 g

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Blood brain BarrierHigh throughput ImagingStereotactic NeuronavigationFocused UltrasoundGliomasNeuro navigated SystemDrug EvaluationPrecision MedicineSonoporationTransducer CalibrationPreclinical StudiesAnesthesia ProtocolMouse Model

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