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>

<ol>
	<li>Choi, J. 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. (19), 30238 (2019).</li><li>Golde, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+questions+for+Alzheimer's+disease+immunotherapy.">Open questions for Alzheimer's disease immunotherapy.</a> Alzheimers Research &amp; Therapy. 6 (1), 3 (2014).</li><li>Nisbet, R. M., 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=Combined+effects+of+scanning+ultrasound+and+a+tau-specific+single+chain+antibody+in+a+tau+transgenic+mouse+model.">Combined effects of scanning ultrasound and a tau-specific single chain antibody in a tau transgenic mouse model.</a> Brain. 140 (5), 1220-1230 (2017).</li><li>Janowicz, P. W., Leinenga, G., G&#246;tz, J., Nisbet, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-mediated+blood-brain+barrier+opening+enhances+delivery+of+therapeutically+relevant+formats+of+a+tau-specific+antibody.">Ultrasound-mediated blood-brain barrier opening enhances delivery of therapeutically relevant formats of a tau-specific antibody.</a> Scientific Reports. 9 (1), 9255 (2019).</li><li>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=Scanning+ultrasound+removes+amyloid-beta+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-beta and restores memory in an Alzheimer's disease mouse model.</a> Science Translational Medicine. 7 (278), 233 (2015).</li><li>Burgess, A., 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=Targeted+delivery+of+neural+stem+cells+to+the+brain+using+MRI-guided+focused+ultrasound+to+disrupt+the+blood-brain+barrier.">Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier.</a> PLoS One. 6 (11), 27877 (2011).</li><li>Chen, H., 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=Focused+ultrasound-enhanced+intranasal+brain+delivery+of+brain-derived+neurotrophic+factor.">Focused ultrasound-enhanced intranasal brain delivery of brain-derived neurotrophic factor.</a> Scientific Reports. 6, 28599 (2016).</li><li>Leinenga, G., Langton, C., Nisbet, R., 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=Ultrasound+treatment+of+neurological+diseases+-+current+and+emerging+applications.">Ultrasound treatment of neurological diseases - current and emerging applications.</a> Nature Reviews Neurology. 12 (3), 161-174 (2016).</li><li>G&#246;tz, J., Halliday, G., Nisbet, R. 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. E., 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=Unilateral+Focused+Ultrasound-Induced+Blood-Brain+Barrier+Opening+Reduces+Phosphorylated+Tau+from+The+rTg4510+Mouse+Model.">Unilateral Focused Ultrasound-Induced Blood-Brain Barrier Opening Reduces Phosphorylated Tau from The rTg4510 Mouse Model.</a> Theranostics. 9 (18), 5396-5411 (2019).</li><li>Valdez, M. A., Fernandez, E., Matsunaga, T., Erickson, R. P., Trouard, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+Diffusion+of+Macromolecule+Delivery+to+the+Brain+via+Focused+Ultrasound+using+Magnetic+Resonance+and+Multispectral+Fluorescence+Imaging.">Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging.</a> Ultrasound in Medicine and Biology. 46 (1), 122-136 (2020).</li></ol>

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>
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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

2.9K 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

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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 ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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 ...

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 ...