We acknowledge support by the Estate of Dr Clem Jones AO, the National Health and Medical Research Council of Australia [GNT1145580, GNT1176326], the Metal Foundation, and the State Government of Queensland (DSITI, Department of Science, Information Technology and Innovation).

All animal experiments were approved by the animal ethics committee of the University of Queensland.
1. In-house microbubble preparation
<ol>
	<li>Weigh out a 9:1 molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt). 0.5 mg of lipid mixture is required per 1 mL of microbubble solution. Alternatively, lipids can be bought already in chloroform, if using pre-dissolved lipids proceed to step 1.3.</li>
	<li>Dissolve the lipid in a small volume of chloroform in a glass beaker.</li>
	<li>Evaporate the chloroform with an evaporator or a nitrogen stream.</li>
	<li>Rehydrate the dried lipid film with 10 mL of PBS + 10% glycerol + 10% propylene glycol solution that has been filtered through 0.22 &#181;m filter.</li>
	<li>Place the rehydrated lipid solution in a water bath sonicator set to 55 &#730;C (above the melting temperature of the lipids) and sonicate until fully dissolved.</li>
	<li>Aliquot lipid solution into autoclaved 1.5 mL HPLC vials and screw on the septa caps.</li>
	<li>Aspirate all of the air in the vial with a 5 mL syringe equipped with a 27 G needle and create a vacuum in the vial.</li>
	<li>Add octofluoropropane to the vial with the included syringe, drawing up the gas from the canister. Fill the vial with 1-2 mL of octafluoropropane by reading the volume in the syringe.</li>
	<li>Seal each vial with paraffin film and refrigerate.</li>
	<li>On the day of the experiment, bring the vial to room temperature, add 0.5 mL of 0.9% NaCl solution to the vial, then place the vial in an amalgamator and agitate for 45 s (preset time) to produce the microbubbles.</li>
</ol>
2. Microbubble quality control using a coulter counter
<ol>
	<li>Take the microbubble solution out of the amalgamator and vent the gas from the vial by piercing the septa with a 19&#160;G needle.</li>
	<li>Dilute the microbubble solution by performing two-step 1:5,000 serial dilutions by adding 100 &#181;L of microbubble solution into 5 mL of filtered flow solution using a 1 ml syringe with 19 G needle, and then taking 100 &#181;L of 1:50 diluted microbubble solution and pipetting into 10 mL filtered flow solution in a cuvette using a pipette.</li>
	<li>Check that the electrolyte tank has sufficient flow solution and that the waste tank is empty.</li>
	<li>Place the cuvette in the coulter counter platform and lock it into place. Use a 30 &#181;m aperture for sample acquisition.</li>
	<li>In the software, load the standard operating method (SOM), then select Edit SOM | concentration. Enter 5000x dilution.</li>
	<li>In the software, choose a suitable file name. For example, microbubble_1_date.</li>
	<li>Load and secure the cuvette into the platform.</li>
	<li>In the software select Run| Preview and verify that the sample concentration is less than 10%. If this number is higher than 10%, perform a new dilution of the microbubbles with a higher dilution factor.</li>
	<li>Select &#39;Start&#39; to begin an acquisition of the sample to obtain the initial readout.</li>
	<li>Rinse the aperture of the Coulter counter with filtered flow solution after each measurement. Repeat steps 2.2-2.9 to obtain 3 replicates.</li>
	<li>Place a cuvette with diluted microbubble solution into a sonicator water bath and sonicate for 30 s.</li>
	<li>Measure the solution with sonicated microbubbles and label as blank.</li>
	<li>In the software, subtract the final readout from the initial readout. This subtracts any particles that are not microbubbles and do not contain gas.</li>
	<li>Select Display Results in the software, to display the microbubble concentration, size distribution, average size, and volume concentration.</li>
</ol>
3. Fluorescent antibody labeling
<ol>
	<li>Obtain a 1 mg/mL solution of mouse IgG in PBS without any additives.</li>
	<li>Label 1 mg of mouse IgG with AlexaFluor 647 in 0.1 M sodium bicarbonate buffer by following the manufacturers&#39; instructions located in the kit. This amount of fluorescently labeled IgG is sufficient to perform this procedure on 5-7 adult mice where a 5 mg/kg dose of antibody is administered.</li>
	<li>Add the dye to the solution of IgG in 0.1 M sodium bicarbonate buffer and incubate for 15 min at room temperature.</li>
	<li>Purify the fluorescently labeled antibody by pipetting the antibody solution into a spin column and centrifuging at 1,000 x g for 5 min. Free dye will remain in the column bed.</li>
	<li>Use a spectrophotometer to measure the protein concentration. Measure the absorbance of the conjugate solution at 280 nm and 650 nm (A280 and A650). Calculate the concentration of protein in the sample using the equation: 
	Protein concentration (M) = [A280 - (A650 x 0.03)] x dilution factor / 203,000.</li>
	<li>Use a spectrophotometer to calculate the degree of labeling using the equation: 
	moles dye per mole protein = A650 x dilution factor / 239 000 x protein concentration (M). 
	NOTE: An acceptable degree of labeling is 3-7 moles dye per mole protein and typically obtained degree of labeling is around 6.</li>
</ol>
4. Ultrasound set-up
<ol>
	<li>Using the focused ultrasound system, add the 5 mm spacer to the water bolus to position the ultrasound focus 9 mm below the bottom of the water bolus.</li>
	<li>Fill the water bolus with approximately 300 mL of deionized water that has been degassed with an inline degasser for 20 min (oxygen content should be below 3 ppm). Place the annular array into the filled water bolus and use a dental mirror to check that there are no air bubbles on the surface. If present on the surface, remove the annular array and replace it in the water bolus.</li>
	<li>Launch the application software. In the waveform menu, select Set waveform duty cycle. Settings are PRF (Hz) 10, duty cycle 10%, focus 80 mm, center frequency 1 MHz, amplitude (MPa peak negative pressure) 0.65 MPa, mechanical index = 0.65. Press Set to define the waveform and store it in memory. 
	NOTE: The focused ultrasound system is pre-calibrated by the manufacturer from measurements taken by a calibrated hydrophone.</li>
	<li>In the focused ultrasound system software, define a treatment plan. This requires defining a treatment region consisting of multiple individual treatments sites, and defining actions to be taken at each of those treatment sites. In this case, the treatment zone is one hemisphere of the mouse brain.</li>
	<li>In the motion controller window, go to the Scan tab and Enter start, stop and increment value for motion in the x dimension, and start, stop and increment value for motion in the y direction. Enter values for X: start -4, stop 3.50 and Y: -3.00, stop 3, Increment 1.5, # loops: 1.</li>
	<li>Define the actions for treatment sites. In the motion controller window, select the Event button. In the Script Editing Window, select a list of actions that will be executed in the order selected at each treatment site. Set Movement Type to raster grid at the top of the script window. In the events tab select Add Actions to move them to the script panel, and add Move Synchronously, Start trigger arb, wait, Stop trigger arb. Click on the wait action and select a wait time of 6, 000 ms. 
	NOTE: These settings will make the treat a 6 x 5 grid of treatment spots spaced 1.5 mm apart, with each spot having a treatment duration of 6 s. The total duration to sonicate a mouse brain is approximately 3 min. This size treatment grid is suitable for adult C57/Bl6 mice weighing approximately 30 g. The size of the grid of treatment spots can be adjusted up or down depending on the size of the mouse.</li>
</ol>
5. Animal preparation
<ol>
	<li>Weigh the mouse with a balance accurate to 0.1g.</li>
	<li>Anesthetize mouse with 90 mg/kg ketamine and 6 mg/kg xylazine intraperitoneally. Test for absence of reflexes with a toe pinch. Alternatively, mice can be anesthetized with isoflurane using an appropriate inhalational anesthetic apparatus with an appropriate face mask. If using isoflurane, the mouse should be placed on a heat pad during the ultrasound to prevent hypothermia.</li>
	<li>Use an electric razor to shave the hair from the head of the animal, then apply hair removal cream with a cotton bud, leave on for 2-3 min or until the hair is wiped away clean with a damp piece of gauze.&#160;Take care that the hair removal cream does not get in the eyes of the mouse.</li>
	<li>Mark the center of the mouse&#8217;s head with a permanent marker. The transducer has a hole in its center and the transducer focus and focal spot can be aligned visually.</li>
	<li>Fill a small weigh boat that has previously had the bottom cut off and replaced with plastic wrap glued to the bottom of the weigh boat with ultrasound gel. This serves as an 8 mm spacer and provides good coupling to the head of the mouse and allows visual inspection of the focus of the transducer aligned with the head of the mouse.</li>
</ol>
6. Microbubble preparation
<ol>
	<li>Warm a microbubble vial to room temperature. To activate, add 0.5 mL of 0.9% NaCl solution to the vial and place in an amalgamator to agitate for 45 s to produce the microbubbles.</li>
	<li>Vent the vial by piercing the septa with a 27 G needle.</li>
</ol>
7. Ultrasound treatment
<ol>
	<li>Invert the vial of microbubbles and gently draw up 1 &#181;L/g bodyweight of the solution. To this add solution of fluorescently labeled antibody and mix gently in the syringe. The maximum volume injected is 150 &#181;L. 
	NOTE: In-house prepared microbubbles are approximately 60-fold less concentrated than the clinically used (e.g., Definity microbubbles). Adjust the volume or concentration such that the number of microbubbles injected are similar to those clinically used (ie. Definity 1.2 x 108 microbubbles/kg body weight).</li>
	<li>Inject the microbubble and antibody solution retroorbitally, taking care to inject gently and slowly.&#160;Then, apply ophthalmic ointment to the mouse&#39;s eyes.</li>
	<li>Place the mouse in the head holder (see Table of Materials) and fix the nose of the mouse. Then place the ultrasound gel-filled small weigh boat on top of the head.</li>
	<li>Lower the water bolus until it sits on top of the ultrasound gel in the weigh boat.</li>
	<li>Use the joystick to move the transducer focus to the center of the head. Select Reset Origin in the motion tab.</li>
	<li>Select complete scan. Steps 7.3-7.6 will take 2 min.</li>
	<li>For consistency, set a timer to ensure a 2 min delay between injecting microbubbles and selecting complete scan.</li>
	<li>After the treatment is complete, apply ophthalmic ointment to the eyes and place mouse in a warmed recovery chamber. If hypothermia is observed during the procedure, a warming pad can be placed under the mouse to provide supplemental heat during the procedure.</li>
</ol>
8. Tissue harvesting and processing
<ol>
	<li>At the timepoint of interest after ultrasound delivery (at least 1 h to detect high levels of antibody in the brain) deeply anaesthetise the mouse with an overdose of Pentobarbitone solution (100 mg/kg) and transcardially perfuse the mouse with 30 mL&#160;PBS. A good perfusion is required to specifically detect fluorescently-labelled antibodies that have been delivered to the brain.</li>
	<li>Collect the brain and fix by immersion in 4% PFA for 24 h at 4 &#176;C, then wash with PBS.</li>
	<li>Image the brain in an infrared scanner by placing the brain on the tray and by acquiring an image in the 700 nm channel. 
	NOTE: After fixation, the brain is removed from the PFA, washed in PBS and sectioned. Alternatively, it can be placed in ethylene glycol cryoprotectant solution for 24 h at 4 &#176;C or until submerged, and then moved to a new cryoprotectant containing vial and placed at -20 &#176;C for long term storage.</li>
	<li>Section the brain cold in PBS using a vibratome. Glue the brain to the platform and cut 30-40 &#181;m sections and collect into PBS. 
	NOTE: Lysosomal autofluorescence (prevalent in sections from animals older than about 12 months) should be bleached by overnight illumination of the sections in a light chamber box, at room temperature and in PBS containing azide (0.02%) to block bacterial growth.</li>
</ol>
9. Tissue staining and image acquisition
<ol>
	<li>Transfer sections to blocking solution (5% BSA in 0.2% Triton/PBS) for 2 h at room temperature, then wash the sections 3x by replacing the solution with 0.2% Triton/PBS.</li>
	<li>Incubate sections at 4 &#176;C overnight with the primary antibodies against Iba1 (dilution 1:1,000) and CD68 (dilution 1:500), in 0.2% Triton/PBS, followed by 3x washes with 0.2% Triton/PBS.</li>
	<li>Incubate sections for 2 h at room temperature with fluorescent secondary antibodies (dilution 1:500), followed by 3x washes with 0.2% Triton/PBS only. 
	NOTE: The nuclei can also be stained using DAPI solution (0.5 &#181;g/mL).</li>
	<li>Transfer the sections to a slide and coverslip with hard-set mounting media. Allow sufficient time for the mounting medium to solidify before visualization and image acquisition (overnight).</li>
	<li>With a confocal microscope obtain z-stack images (at least 10 &#181;m depth and 0.3 &#181;m step size, 10 images per animal) of the ultrasound targeted brain area by using a confocal microscope and an objective of at least 40x magnification. 
	NOTE: Be careful to acquire images within the signal dynamic range, avoiding under-/over-exposure. Save the image files in the corresponding software format used by the microscope.</li>
</ol>
10. Image analysis
<ol>
	<li>Convert the z-stack microscopy files using the image analysis software file importer.</li>
	<li>Open the converted files into the software and adjust the intensity of the Iba1 channel to observe microglial cells.</li>
	<li>Crop a single microglial cell by drawing a box around it, select &#8216;crop&#8217; and save the new file.</li>
	<li>Build the 3D surface rendering of the IBA1 signal by:
	<ol>
		<li>Open the single cell Imaris file generated in the previous step.</li>
		<li>Add a surface to the file.</li>
		<li>Select the channel corresponding to the Iba1 staining.</li>
		<li>Apply a threshold that should overlap the Iba1 staining</li>
		<li>Select only the structure of interest that is forming the microglial cell of interest</li>
	</ol>
	</li>
	<li>Finalize the process
	<ol>
		<li>Obtain and record the volume of the Iba1 staining</li>
		<li>Build the 3D surface rendering of the CD68 staining</li>
		<li>Inside the IBA1 surface subfile, mask the CD68 channel and remove the voxels outside of the IBA1 staining</li>
		<li>Add a new surface to the file</li>
		<li>Select the channel corresponding to the CD68 staining</li>
		<li>Apply a threshold that should overlap the CD68 staining and is as closely as possible matching the staining volumes</li>
		<li>Finalize the process, as only the intra-microglial CD68 structures will be present in this file and, thus, they do not require another threshold filtering step</li>
		<li>Obtain and record the number and average volume of the CD68 positive structures</li>
		<li>Calculate the relative volume of CD68 structures per its corresponding IBA1 structures as a measure of microglial activation in a phagocytic state.</li>
	</ol>
	</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+and+transient+blood-brain+barrier+opening+in+the+hippocampus+of+Alzheimer's+double+transgenic+mice+using+focused+ultrasound.">Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer's double transgenic mice using focused ultrasound.</a> Ultrasonic Imaging. 30 (3), 189-200 (2008).</li><li>Lipsman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+opening+in+Alzheimer's+disease+using+MR-guided+focused+ultrasound.">Blood-brain barrier opening in Alzheimer's disease using MR-guided focused ultrasound.</a> Nature Communications. 9 (1), 2336 (2018).</li><li>Pandit, R., Chen, L., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+physiology+and+strategies+for+drug+delivery.">The blood-brain barrier: physiology and strategies for drug delivery.</a> Advanced Drug Delivery Reviews. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Pathogenesis+of+the+Tauopathies.">Molecular Pathogenesis of the Tauopathies.</a> Annual Reviews of Pathology. 14, 239-261 (2019).</li><li>Pandit, R., Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+ultrasound+treatment+of+tau+transgenic+mice+clears+neuronal+tau+by+autophagy+and+improves+behavioral+functions.">Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.</a> Theranostics. 9 (13), 3754-3767 (2019).</li><li>Karakatsani, M. 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We have nothing to disclose.

Fluorescently-labeled antibodies can be delivered to the brain using focused ultrasound together with microbubbles applied in a scanning mode. Antibody delivery, microglial morphology and lysosomal enlargement can be detected by fluorescence microscopy following scanning ultrasound. Microglia can take up into their lysosomes antibodies and antigens that the antibodies have bound to in an Fc-receptor-mediated process4.
There are a number of critical steps to achieve repe...

Therapeutic ultrasound is an emerging technology aimed at treating brain diseases in a noninvasive manner, in part by facilitating access of therapeutic agents to the brain1,2,3. As only a small fraction of therapeutic antibodies targeting brain diseases are taken up by and retained in the brain4, therapeutic ultrasound offers the possibility to increase their uptake and target engagement5,6.
In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the blood-brain barrier (BBB) using microbubbles. To achieve this, ultrasound is applied through the skull into the brain in multiple spots using a scanning mode we refer to as scanning ultrasound (SUS)7. The mechanical interaction between the ultrasound energy, the intravenously injected microbubbles and the brain vasculature transiently separates the tight junctions of the BBB in a given sonication volume, allowing antibodies and other cargoes including therapeutic agents to effectively cross this barrier7,8,9. Moreover, ultrasound has been shown to facilitate the uptake of antibodies from the interstitial brain into brain cells, such as neurons, where the antibody distributes throughout the cell body and even into neuritic processes5,10.
Alzheimer&#39;s disease is characterized by an amyloid-&#946; and tau pathology11, and a host of animal models is available to dissect pathogenic mechanisms and validate therapeutic strategies. A&#160;SUS approach, by which ultrasound is applied in a sequential pattern across the entire brain, when repeated over several treatment sessions, can reduce amyloid plaque pathology in the brains of amyloid-&#946;-depositing amyloid precursor protein (APP) mutant mice and activate microglia which take up the amyloid, leading to improvement in cognitive function7. BBB opening with ultrasound and microbubbles also reduces tau pathology in pR5, K3 and rTg4510 tau transgenic mice5,12,13. Importantly, whilst microglia remove extracellular protein deposits, one of the underlying clearance mechanisms for intraneuronal pathologies induced by SUS is the activation of neuronal autophagy12.
Here, we outline an experimental process, by which fluorescently labeled antibodies are prepared, and then mixed with in-house lipid-based microbubbles, followed by retroorbital injection into anesthetized mice. Retroorbital injection is an alternative to tail vein injection which we have found to be equally efficacious and simpler to repeatedly perform. This is immediately followed by applying SUS to the brain. To determine the therapeutic antibody uptake, mice are sacrificed and the increased antibody concentration in the brain is then quantified. As a proxy of the change in brain homeostasis, microglial phagocytic activity is determined by histology and volumetric 3D reconstruction.
The generated data suggest that ultrasound delivery of antibodies is a potentially attractive approach to treat neurodegenerative diseases. The protocol can be similarly applied to other drug candidates, as well as model cargos such as fluorescently labeled dextrans of defined sizes14.

<table><tbody><tr><td>1,2-distearoyl-sn-glycero-3-phosphocholine</td><td>Avanti</td><td>850365C</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000]</td><td>Avanti</td><td>880128C</td><td></td></tr><tr><td>AlexaFluor 647 antibody labeling kit</td><td>Thermo Fisher</td><td>A20186</td><td></td></tr><tr><td>CD68 antibody</td><td>AbD Serotec</td><td>MCA1957GA</td><td>Use 1:1000 dilution</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>372978</td><td></td></tr><tr><td>Coulter Counter (Multisizer 4e)</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Thermo FIsher</td><td>A-11008</td><td>Use 1:500 dilution</td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Thermo Fisher</td><td>A-11077</td><td>Use 1:500 dilution</td></tr><tr><td>head holder (model SG-4N, Narishige Japan)</td><td></td><td></td><td></td></tr><tr><td>Iba1 antibody</td><td>Wako</td><td>019-19741</td><td>Use 1:1000 dilution</td></tr><tr><td>Image analysis software</td><td>Beckman Coulter</td><td>#8547008</td><td></td></tr><tr><td>Isoflow flow solution</td><td>Beckman Coulter</td><td>B43905</td><td></td></tr><tr><td>Near infrared imaging system Odyssey Fc</td><td>Licor</td><td>2800-03</td><td></td></tr><tr><td>Octafluoropropane</td><td>Arcadophta</td><td>0229NC</td><td></td></tr><tr><td>Propylene Glycol</td><td>Sigma-Aldrich</td><td>P4347</td><td></td></tr><tr><td>TIPS (Therapy Imaging Probe System)</td><td>Philips Research</td><td>TIPS_007</td><td></td></tr><tr><td></td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

delivery of antibodies into the brain using focused scanning ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Using this protocol fluorescently-labeled antibodies are delivered to the brain and can be detected, along with microglia activation. The conclusion that can be drawn is the use of focused ultrasound and microbubbles markedly enhances brain uptake of antibodies and can deliver antibodies to the whole brain or hemisphere of a mouse when used in a scanning mode. Figure 1 shows the TIPS ultrasound application device (different components labeled) that is used to open the BBB. <strong class="xfi...

Watch this Scientific Journal Video about Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound at JoVE.com

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

delivery-antibodies-into-brain-using-focused-scanning

Research

JoVE Journal

Neuroscience

3.0K Views.  The University of Queensland. Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Video: Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

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

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

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

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

Medicine

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Bioengineering

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Convection-Enhanced Delivery of Antibodies into a Mouse Brain

Source: Beffinger, M., et al. <a href="https://app.jove.com/v/59675">Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.</a> J. Vis. Exp. (2019).
This video demonstrates a convection-enhanced delivery (CED) technique for antibody delivery into the mouse brain. The skull of an anesthetized mouse is exposed, and a burr hole is created at the target area for antibody delivery. A step catheter containing antibodies is inserted at the target site, and via an infusion pump, a pressure gradient is created to deliver antibodies at a controlled rate.

Konwekcyjne dostarczanie przeciwciał do mózgu myszy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

An Ultrasound-Assisted Delivery of Antibodies in a Mouse Model

Source: Leinenga, G. et al., <a href="https://app.jove.com/v/61372">Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates a procedure of ultrasound-assisted antibody delivery in a mouse model. Retro-orbitally injected microbubbles oscillate under ultrasound, creating a transient disruption in the blood-brain barrier, permitting labeled antibodies to enter the brain.

Wspomagane ultradźwiękami dostarczanie przeciwciał w modelu mysim

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

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

Focus Ultrasounds Mikropęcherzykowe otwieranie bariery krew-mózg: technika tworzenia zlokalizowanych przejściowych otworów w barierze krew-mózg myszy za pomocą sonoporacji

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