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 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&#160;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.
<ol>
	<li>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.</li>
	<li>Place the transducer in the 3D-printed cone.</li>
	<li>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.</li>
	<li>Mount the transducer above the animal on a motorized linear stage as shown in Figure 1 allowing automatic vertical positioning of the transducer.</li>
	<li>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 (&#60; 0.1 mm) of the animal under the beam.</li>
	<li>Connect the transducer to the acoustic emission chain shown in Figure 1 consisting of a transducer, a function generator and a power amplifier.</li>
	<li>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.</li>
	<li>Calibrate the system and determine the focus point of the transducer in correspondence to vertical positioning of the animal on the stereotactic platform.</li>
</ol>
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.
<ol>
	<li>Allow the animal to acclimatize for at least one week in the animal facility and weigh the animal regularly.</li>
	<li>Administer buprenorphine (0.05 mg/kg) via subcutaneous (s.c.) injection 30 min prior to FUS treatment to start analgesic treatment.</li>
	<li>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.</li>
	<li>Apply eye ointment to prevent dry eyes and avoid possible injury.</li>
	<li>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.</li>
	<li>For experiments with BLI tumor models, inject 150 &#181;L of D-luciferin (30 mg/mL) intraperitoneal (i.p.) with a 29 G insulin syringe for BLI image-guidance.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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 &#956;m and more than 90% of the bubbles are smaller than 8 &#956;m.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Shield the animal&#8217;s nostrils and mouth with adhesive tape to prevent ultrasound gel interfering with breathing.</li>
	<li>Apply ultrasound gel on top of the animal&#8217;s head.</li>
	<li>Retract the skin of the animals&#8217; neck, lubricate the needle hydrophone with ultrasound gel and place the needle hydrophone in the direct vicinity of the occipital bone.</li>
	<li>Guide the transducer to the correct position using the image-processing pipeline and the focus point.</li>
	<li>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.</li>
	<li>Activate microbubbles as described by the manufacturer. Inject one bolus of 120 &#181;L (5.4 &#181;g) of microbubbles.</li>
	<li>Flush the tail vein catheter with saline to check the opening of the catheter.</li>
	<li>Inject the microbubbles and start the insonation.</li>
	<li>Record microbubble cavitation with the needle hydrophone.</li>
	<li>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.</li>
	<li>Monitor the animal until the predetermined time point or before the humane endpoint.</li>
</ol>
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 &#181;m (80% of the bubbles below 8 &#181;m) excited with a burst-tone pulse of 10 ms duration at a frequency of 1 MHz, as originally suggested by McDannold et al.12.
<ol>
	<li>Fourier-transform the recorded PCD signal from the time-domain into the frequency domain.</li>
	<li>Integrate the resulting spectral power for stable cavitation detection around the 2nd and 3rd harmonic (&#177; 50 kHz), as shown in Figure 3 (green box at 2 and 3 MHz).</li>
	<li>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 (&#177; 150 kHz), as shown in Figure 3 (red boxes).</li>
	<li>Integrate the spectral power around the principle frequency (1 MHz &#177; 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.</li>
</ol>

The authors have nothing to disclose.

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 ...

a-high-throughput-image-guided-stereotactic-neuronavigation-focused

Research

JoVE Journal

Neuroscience

4.7K Views.  Cancer Center Amsterdam. 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.

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

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.

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 challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

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 between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

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

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

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

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

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

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.

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 delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

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 useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

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 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.

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

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In vivo Electroporation of Developing Mouse Retina

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

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

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

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

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

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

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

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

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