This study was partially supported by the National Institute of Dental and Craniofacial Research (NIDCR) Grant K99DE032397, UCSF Department Seed Grant (No. 7504831), UCSF RAP grant (PI: Kazim Narsinh, MD), and the German Research Foundation (DFG, GA 3535/1-1).

All animal experiments in this study were approved by the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley rats (200-250 g) were used in this study. Rats were housed and cared for according to the National Institutes of Health Office of Animal Care and Use Guidelines in mice and rats. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Animal preparation
NOTE: For in-house procedures, the animals were transported to the preparation room and the procedures were conducted as follows:
<ol>
	<li>Place the rats individually in a Lucite chamber and expose isoflurane (2%-3%) mixed with oxygen extracted from a medical oxygen tank until they are unconscious (following institutionally approved protocols). 
	NOTE: Anesthesia using isoflurane enables the implementation of a long-term MRI protocol.</li>
	<li>Remove the rats from the chamber and shave hair from the anterior and posterior areas of the neck using a hair removal (depilatory) cream.</li>
	<li>Apply lubricant ointment to the eyes.</li>
	<li>Place the nose into the nose cone to maintain anesthesia with spontaneous respiration (isoflurane vaporizer between 2%-2.5%, oxygen flow meter to 1-1.5 L/min).</li>
	<li>Place the anesthetized rat onto a heated surgical table and confirm with a toe pinch. 
	NOTE: The MRI protocol included contrast-enhanced sequences, such as T1-weighted gradient echo imaging and T2-weighted spin echo imaging. These sequences were conducted both before and after injection of gadolinium (GAD)-based contrast agents to assess the blood-brain barrier (BBB) permeability. This involves administering GAD contrast agents while the rat is in an MRI scanner7. To facilitate this process, catheter tubing extending from the catheter cannulated in the rat was used. The extension included a catheter connector and polyethylene tubing measuring 30 cm. To ensure patency and prevent leakage, the extension tubing was flushed with heparinized glycerol (250 IU heparin/1 mL glycerol).</li>
</ol>
2. Stereotactic-guided focused ultrasound system
NOTE: The animals were transferred and placed on a table in a stereotactic FUS system. The rat&#39;s nose was placed into the nose cone to maintain anesthesia. The FUS system comprises a function generator, an RF amplifier, and an oscilloscope. Before setting up the transducer, we ensured the proper connection of all devices with the system. The transducer setup was completed using the following steps:
<ol>
	<li>Place a sharp pointer at the position of the Bregma in the skull and save the position.The position of Bregma can be identified by pointing the location of 9.0 mm anterior to Lambda on the midline8. The Lambda can be found at the center of the line between bilateral ear drums9.</li>
	<li>Place ultrasound gel on the rat&#39;s head, water coupling bag, and the transducer (1 MHz frequency). Ensure that no air bubbles are present between the rat&#39;s skin, the water coupling bag, and the transducer.</li>
	<li>Connect a coaxial BNC cable of the transducer to the input of the FUS system module.</li>
	<li>In a control computer) Click on load for pre-registered rat images; a number of focal spots and acoustic pressure are chosen.</li>
	<li>In a control computer) Click on the motion test in the treatment moduleto ensure that the transducer can shift between spots within a burst period.</li>
	<li>Set up the sonication parameters described in step 3.</li>
</ol>
3. Microbubble infusion and sonication
<ol>
	<li>Activate the microbubble. 
	NOTE: The microbubble used in this study was characterized by a diameter of 1.7 &#181;m and a concentration of 0.2 &#215; 109 particles per milliliter10. The microbubble vial was activated based on the prescribing instructions provided by the vendor and a previously published report11.
	<ol>
		<li>Prepare a 5 mL syringe with a luer lock tip and filled with 5 mL of additive-free 0.9% sodium chloride injection.</li>
		<li>Remove the green cap, and connect the syringe to the vented dispensing pin (see Table of Materials) by screwing it clockwise.</li>
		<li>Remove the pin protection and place it in the center of the vial&#39;s rubber stopper. Press down firmly until the spike is fully inserted into the stopper.</li>
		<li>Empty the entire 5 mL syringe into the vial by pushing on the plunger rod.</li>
		<li>Shake the vial vigorously for 20 s to mix all the contents. A consistent white, milky liquid will show that sulfur hexafluoride lipid microspheres have formed.</li>
		<li>To obtain the required dose, invert the syringe, and slowly withdraw the intended volume of the suspension into a 5 mL syringe.</li>
	</ol>
	</li>
	<li>Use an infusion pump. 
	NOTE: The infusion approach12 was used to deliver microbubbles to rats using a syringe pump. A syringe containing microbubbles was connected to a catheter connector inserted into the rat. Microbubbles were transferred through polyethylene tubing, and the infusion pump flow rate was set to 0.66 mL/min. This infusion rate allows the injection of 2 mL of microbubbles from a 3 mL syringe over 3 min. The infusion started before sonication for 1 min and continued throughout the 2 min sonication period.</li>
	<li>Perform sonication. 
	NOTE: In this study, FUS pulses were chosen with the following parameters: (1) Target region: left hippocampus; (2) Number of spots: 6 to cover the hippocampus in the brain; (3) Burst length: 10 ms; (4) Burst period: 1200 ms; (5) Acoustic pressure: 0.65 MPa; (6) &#39;Burst mode&#39; in the sonication mode menu in the FUS system; (7) Sonication time: 120 s.</li>
</ol>
4. MRI procedure using a 3T preclinical scanner
<ol>
	<li>After sonication, transfer the animals to a table in a preclinical 3T cryogen-free scanner.</li>
	<li>Position the rats in the prone position. Maintain the temperature of the heating pad in the scanner, and monitor the breathing rate using a pneumatic pillow sensor. Perform the MRI protocol to test various types of MR imaging (Table 1).</li>
	<li>Include other quantitative imaging techniques, such as diffusion-weighted imaging (DWI), T2* mapping, T1 mapping, and dynamic contrast-enhanced (DCE) MRI, in addition to contrast-enhanced MRI, to demonstrate its potential practicality for diverse imaging studies. Refer to Table 1 for the MRI protocols used in this study.</li>
	<li>Inject GAD-based MRI contrast agents (Gadavist, 1 mmol/kg) after the T1 weighted MR gradient echo sequence, and acquire post-GAD images. 
	NOTE: For a 200 g rat, we injected 200 &#181;L of the MRI contrast agent and followed it with a 200 &#181;L saline solution to flush the tubing.</li>
</ol>
5. Macroscopic examination of the rat brain
NOTE: After the MRI scan, 2 mL of Evans blue solution (2% diluted with phosphate-buffered saline) was injected, and the rat was allowed to rest for at least 30 min to allow the solution to spread and circulate throughout the body. Subsequently, the rats were euthanized using an overdose of isoflurane (5%) for macroscopic examination of the brain (following institutionally approved protocols). To confirm euthanasia, toe punching was required and the animal was observed for cessation of breathing for at least 1 min. The perfusion procedure was performed, followed by an examination of the rat brain. The perfusion procedure is summarized as follows:
<ol>
	<li>Perform a thoracotomy using a surgery scissor to expose organs in the chest and visualize the cardiac region.</li>
	<li>Incise (~5 mm) the right atrium.</li>
	<li>Introduce saline solution into the left ventricle for perfusion purposes.</li>
	<li>Open the skull bone with a surgical scissors to unveil and present the brain. 
	NOTE: Adequate perfusion should result in a uniformly bright appearance of the brain, while persisting blue areas indicate BBB-associated albumin leakage, as detected by Evans blue.</li>
</ol>

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

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	<li>Daneman, R., Prat, A. <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.">The Blood-Brain Barrier.</a> Cold Spring Harb Perspect Biol. 7 (1), a020412 (2015).</li><li>Meng, Y., 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=Applications+of+focused+ultrasound+in+the+brain:+from+thermoablation+to+drug+delivery.">Applications of focused ultrasound in the brain: from thermoablation to drug delivery.</a> Nat Rev Neurol. 17 (1), 7-22 (2021).</li><li>Mungur, R., 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=Low-intensity+focused+ultrasound+technique+in+glioblastoma+multiforme+treatment.">Low-intensity focused ultrasound technique in glioblastoma multiforme treatment.</a> Frontiers in Oncology. 12, 903059 (2022).</li><li>Boado, R. 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=Pharmacokinetics+and+brain+uptake+of+a+genetically+engineered+bifunctional+fusion+antibody+targeting+the+mouse+transferrin+receptor.">Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor.</a> Mol Pharm. 7 (1), 237-244 (2010).</li><li>Hendricks, B. K., 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=Novel+delivery+methods+bypassing+the+blood-brain+and+blood-tumor+barriers.">Novel delivery methods bypassing the blood-brain and blood-tumor barriers.</a> Neurosurgical Focus. 38 (3), E10 (2015).</li><li>Hynynen, K., 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=Non-invasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Non-invasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220 (3), 640-646 (2001).</li><li>Hynynen, K., 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=Local+and+reversible+blood-brain+barrier+disruption+by+non-invasive+focused+ultrasound+at+frequencies+suitable+for+trans-skull+sonications.">Local and reversible blood-brain barrier disruption by non-invasive focused ultrasound at frequencies suitable for trans-skull sonications.</a> NeuroImage. 24 (1), 12-20 (2005).</li><li>Yao, X., 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=Non-invasive,+targeted+creation+of+neuromyelitis+optica+pathology+in+AQP4-IgG+seropositive+rats+by+pulsed+focused+ultrasound.">Non-invasive, targeted creation of neuromyelitis optica pathology in AQP4-IgG seropositive rats by pulsed focused ultrasound.</a> J Neuropathol Exp Neurol. 78 (1), 47-56 (2019).</li><li>Cecyn, M. N., Abrahao, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+do+you+measure+the+Bregma+for+rodent+stereotaxic+surgery.">Where do you measure the Bregma for rodent stereotaxic surgery.</a> IBRO Neurosci Rep. 15, 143-148 (2023).</li><li>Kotopoulis, S., 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=SonoVue+vs.+Sonazoid+vs.+Optison:+Which+bubble+is+best+for+low-intensity+sonoporation+of+pancreatic+ductal+adenocarcinoma.">SonoVue vs. Sonazoid vs. Optison: Which bubble is best for low-intensity sonoporation of pancreatic ductal adenocarcinoma.</a> Pharmaceutics. 14 (1), 98 (2022).</li><li>Bracco Diagnostics Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LUMASON (sulfur hexafluoride lipid-type A microspheres) for injectable suspension, for intravenous use or intravesical use. , (2016).</li><li>Lapin, N. 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=Consistent+opening+of+the+blood+brain+barrier+using+focused+ultrasound+with+constant+intravenous+infusion+of+microbubble+agent.">Consistent opening of the blood brain barrier using focused ultrasound with constant intravenous infusion of microbubble agent.</a> Sci Rep. 10, 16546 (2020).</li><li>Vlachos, F., 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=Permeability+dependence+study+of+the+focused+ultrasound-induced+blood-brain+barrier+opening+at+distinct+pressures+and+microbubble+diameters+using+DCE-MRI.">Permeability dependence study of the focused ultrasound-induced blood-brain barrier opening at distinct pressures and microbubble diameters using DCE-MRI.</a> Magn Reson Med. 66 (3), 821-830 (2011).</li><li>Singh, 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=An+all-ultrasound+cranial+imaging+method+to+establish+the+relationship+between+cranial+FUS+incidence+angle+and+transcranial+attenuation+in+non-human+primates+in+3D.">An all-ultrasound cranial imaging method to establish the relationship between cranial FUS incidence angle and transcranial attenuation in non-human primates in 3D.</a> Sci Rep. 14 (1), 1488 (2024).</li><li>Plaksin, 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=Magnetic+resonance+imaging+analysis+predicts+nanoparticle+concentration+delivered+to+the+brain+parenchyma.">Magnetic resonance imaging analysis predicts nanoparticle concentration delivered to the brain parenchyma.</a> Commun Biol. 5 (1), 1-11 (2022).</li><li>Singh, 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=Guiding+and+monitoring+focused+ultrasound+mediated+blood-brain+barrier+opening+in+rats+using+power+Doppler+imaging+and+passive+acoustic+mapping.">Guiding and monitoring focused ultrasound mediated blood-brain barrier opening in rats using power Doppler imaging and passive acoustic mapping.</a> Sci Rep. 12 (1), 14758 (2022).</li></ol>

The authors have no relevant conflicts of interest to declare.

A protocol and animal procedure using a rat model of jugular vein catheterization for BBB opening studies has been developed. While numerous preclinical protocols exist, this protocol incorporates a commercially available FUS system and a rat model, minimizing animal preparation procedures and thereby ensuring experimental reproducibility.
In this protocol, microbubble selection was based on its stability and room temperature storage. Other commercial microbubbles may be considered as alternat...

The BBB regulates the movement of ions, molecules, and cells between the blood vessels and brain tissues1. This function allows the maintenance of neuronal function and health while preventing the delivery of therapeutic macromolecular agents to the brain tissues. The limited permeability of the BBB can result in decreasing the efficacy and effectiveness of treatments for brain tumors and neurodegenerative diseases, such as Parkinson&#39;s disease, amyotrophic lateral sclerosis, and Alzheimer&#39;s disease2. Hence, various approaches, such as convection-enhanced delivery, direct intracranial injections, osmotic disruption, and enhanced drug design, have been investigated to bypass the BBB for controlled delivery of therapeutic agents3,4,5.
However, these approaches have intrinsic limitations such as invasiveness, risk of infection, risk of hemorrhage, heterogeneity, and rapid efflux of drugs. Low-intensity pulsed focused ultrasound (FUS) with the injection of microbubbles is a promising non-invasive technique allowing localized and reversible BBB disruption. The mechanical effects of ultrasound coupled with microbubbles can transiently alter the permeability of the BBB (i.e., BBB opening)6,7. For further advancements in treatment applications utilizing BBB opening, in vivo small-animal experiments are essential.
In this study, we introduced MRI protocols and animal procedures to monitor BBB opening in a rat model. The protocol included a rat model with a Jugular vein catheter cannulated by a commercial organization (see Table of Materials). A rat model of jugular vein catheterization has the potential benefits of rapid medication administration, hemodynamic monitoring tests, and stable catheterization. BBB opening was accomplished using a preclinical FUS system. Specifically designed for BBB opening in small animal models such as rats and mice, this system incorporates essential components, such as a benchtop system, control computer, transducer, water degasser, and accompanying accessories. The use of this stereotactic-guided preclinical FUS system not only enhances precision, but also streamlines animal procedures and MRI protocols, contributing to the reproducibility and simplicity of the overall experimental protocol.

<table><tbody><tr><td>1 mL syringe</td><td>BD plastipak</td><td></td><td></td></tr><tr><td>20 mL, Sodium Chloride 0.9%</td><td>Hospira, Inc</td><td>00409-7101-67</td><td></td></tr><tr><td>3 mL syringe</td><td>BD plastipak</td><td></td><td></td></tr><tr><td>5 mL syringe</td><td>BD plastipak</td><td></td><td></td></tr><tr><td>Blunt Needle, 21 G</td><td>SAI infusion technologies</td><td>B21-50</td><td></td></tr><tr><td>Catheter connector, 21 G</td><td>SAI infusion technologies</td><td>CC-25-25</td><td></td></tr><tr><td>Catheter lock solution</td><td>SAI infusion technologies</td><td>Sku # HGS-10</td><td>(500 &amp;micro;/mL) Glycerol</td></tr><tr><td>Evans Blue</td><td>Sigma aldrich</td><td>E2129-10g</td><td></td></tr><tr><td>ExAblate Neuro</td><td>Insightec Inc., Israel</td><td></td><td></td></tr><tr><td>Gadavist</td><td>Bayer</td><td>2068062</td><td></td></tr><tr><td>GE&amp;nbsp; isoflurane Vaporizer&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>IPS-12RS Syringe Pump</td><td>Inovenso</td><td>IPS-12</td><td></td></tr><tr><td>isoflurane USP</td><td>Vet one</td><td>501017</td><td></td></tr><tr><td>Lumason microbbuble</td><td>Bracco Diagnostics Inc</td><td>SKJ709700</td><td></td></tr><tr><td>Male Sprague-Dawley rats (200&amp;ndash;250 g)&amp;nbsp;</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Needle, 18 G</td><td>BD Precisionglide</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Mediatech</td><td>21-030-CVR</td><td></td></tr><tr><td>Polyethylene Tubing</td><td>BD Intramedic</td><td></td><td></td></tr><tr><td>preclinical 3T cryogen-free Bruker Biospin scanner&amp;nbsp;</td><td>Bruker</td><td>Biospec3T</td><td></td></tr><tr><td>Rat with jugular vein catheterization</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>RK-50&amp;nbsp; focused ultrasound transducer</td><td>FUS Instruments</td><td>Tx-50-1000-0</td><td></td></tr><tr><td>RK-50 benchtop focused ultrasound system</td><td>FUS Instruments</td><td></td><td></td></tr><tr><td>Sonic Concepts</td><td>Sonic Concepts Inc., WA, USA</td><td></td><td></td></tr><tr><td>vented dispensing pin&amp;nbsp;</td><td>B Braun</td><td>4550560</td><td></td></tr></tbody></table>

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 presents a preclinical protocol for monitoring BBB opening in a rat model using a commercial animal FUS system. The FUS system was set up on a bench or large cart carrier (Figure 1). Rats were anesthetized with isoflurane mixed with oxygen through the nose cone in the frame with the transducer holder. A hydrophone resonating at 500 kHz for detecting acoustic cavitation was inserted into the FUS transducer with a resonant frequency of 1.0 MHz.
As shown i...

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

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 ...

monitoring-blood-brain-barrier-opening-rats-with-preclinical-focused

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

470 Views.  University of California, San Francisco. 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.

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

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

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

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

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

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

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

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

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

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

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

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

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

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

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 ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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.

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

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Chapters in this video

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

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

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

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

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

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

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

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

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