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

Summary

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.

Abstract

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.

Introduction

Despite the many discoveries in basic neuroscience, the number of emerging treatments for neurodevelopmental and neurodegenerative disorders remains relatively limited^1,2. A deeper understanding of the genes, molecules and cellular circuitry involved in neurological disorders has suggested promising treatments unrealizable in humans with current techniques³. Effective treatments are often limited by the need to be brain penetrable and site-specific^4,5,6,7....

Protocol

All experimental procedures were done in accordance with UAB Institutional Animal Care and Use Committee (IACUC) guidelines.

1. Focused ultrasound driving equipment setup

1. Use 50 Ohm coaxial BNC cables to connect (1) the input of the ultrasound transducer to the output of the RF amplifier and (2) the input of the RF amplifier to the output of the function generator.
2. Set the function generator mode to a sinusoid burst once per second with a 1% duty cycle.
   1. For the damped 1 MHz low-power immersion transducer with a 0.8 inch focal distance used with a 50 dB RF amplifier, set the starting settings to: 1 MHz sine ....

Results

Here, we demonstrate that focused ultrasound with microbubbles can induce localized BBB opening using the parameters specified above with both the low-power immersion transducer (Figure 3) and the FUS transducer (Figure 4). First, in early experiments, the low-power immersion transducer was targeted to one brain hemisphere either anterior (Figure 3b) or medial (Figure 3a). Animals were then sacrificed 2.......

Discussion

Here we described a benchtop approach to microbubble assisted FUS BBB opening with alternative approaches including, two different transducers and methods for intracranial targeting with and without MRI guidance. Currently, in order to establish MRI-guided FUS BBB opening in the lab, there is the option to purchase excellent ready-to use devices that provide highly standardized and reproducible results with user-friendly interfaces. However, many labs are not prepared for the cost of such instruments. Therefore, the main.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bubble shaker	Lantheus Medical Imaging	VMIX	VIALMIX, actiation device used to activate Definity microbubbles
Catheter plug/ Injection cap	SAI infusion technologies	Part Number: IC	Catheter plug/ Injection cap
Evans blue dye	Sigma	E2129-10G	Evans blue dye
Function generator	Tektronix	AFG3022B	Dual channel, 250MS/s, 25MHz
FUS transducer, 1.1MHz	FUS Instruments	TX-110	1 MHz MRI-compatible spherically focused ultrasound transducer with a hydrophone
Heating pad for Mice and Rats	Kent Scientific	PS-03	Heating pad- PhysioSuite for Mice and Rats
Infusion pump	KD Scientific	780100	KDS 100 Legacy Single Syringe Infusion Pump
Kapton tape	Gizmo Dorks	https://www.amazon.com/dp/B01N1GGKRC/ ref=cm_sw_em_r_mt_dp_U_GbR7Db56HKD91	Gizmo Dorks Kapton Tape (Polyimide) for 3D Printers and Printing, 8 x 8 inches, 10 Sheets per Pack
Low power immersion transducer, 1MHz	Olympus	V303-SU	Immersion Transducer, 1 MHz, 0.50 in. Element Diameter, Standard Case Style, Straight UHF Connector, F=0.80IN PTF
Magnet sets	WINOMO	https://www.amazon.com/dp/B01DJZQJBG/ ref=cm_sw_em_r_mt_dp_U_JYQ7DbM32E5QC	WINOMO 15mm Sew In Magnetic Bag Clasps for Sewing Scrapbooking - 10 Sets
RF amplifier	E&I	A075	75W
Tail vein catheter	BD	382512/ Fisher Item: NC1228513	24g BD Insyte Autoguard shielded IV catheters (non-winged)
Ultrasound contrast microbubbles	Lantheus Medical Imaging	DE4, DE16	DEFINITY (Perflutren Lipid Microsphere)
Ultrasound gel	Aquasonic	https://www.amazon.com/dp/B07FPQDM4F/ ref=cm_sw_em_r_mt_dp_U_D6Q7Db3J9QP7P	Ultrasound Gel Aquasonic 100 Transmission 1 Liter Squeeze Bottle
Winged infusion sets, 22ga.	Fisher Healthcare	22-258087	Terumo Surflo Winged Infusion Sets
motor controller software	N/A	N/A	custom software written in LabView for controlling the Velmex motor controller
runtime environment for the motor controller software	National Instruments	LabView runtime engine version 2017 or better	https://www.ni.com/en-us/support/downloads/software-products/download.labview.html
3 axis Linear stage actuator (XYZ positioner)	Velmex
bolts	Velmex	MB-1	BiSlide Bolt 1/4-20x3/4" Socket cap screw (10 pack), Qty:3
motor controller	Velmex	VXM-3	Control,3 axis programmable stepping motor control, Qty:1
mounting cleats	Velmex	MC-2	Cleat, 2 hole BiSlide, Qty:6
mounting cleats	Velmex	MC-2	Cleat, 2 hole BiSlide, Qty:2
usb to serial converter	Velmex	VXM-USB-RS232	USB to RS232 Serial Communication Cable 10ft, Qty:1
x-axis linear stage	Velmex	MN10-0100-M02-21	BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1
x-axis stepper motor	Velmex	PK266-03A-P1	Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1
y-axis linear stage	Velmex	MN10-0100-M02-21	BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1
y-axis stepper motor	Velmex	PK266-03A-P1	Vexta Type 23T2, Single Shaft Stepper Motor, Qty:1
z-axis damper	Velmex	D6CL-6.3F	D6CL Damper for Type 23 Double Shaft Stepper Motor, Qty:1
z-axis linear stage	Velmex	MN10-0100-M02-21	BiSlide, travel=10 inch, 2 mm/rev, limits, NEMA 23, Qty:1
z-axis stepper motor	Velmex	PK266-03B-P2	Vexta Type 23T2, Double Shaft Stepper Motor, Qty:1
3D printable files
Immersion transducer mount and pointer			https://www.tinkercad.com/things/cRgTthGXSRq
Stereotaxic frame			https://www.tinkercad.com/things/ilynoQcdqlH
Stereotaxic frame holder			https://www.tinkercad.com/things/aZNgqhBOHAX
9.4T small bore animal MRI	Bruker	Bruker BioSpec 94/20	ParaVision version 5.1
AAV9-hsyn-GFP	Addgene
Cream hair remover	Church & Dwight		Nair cream
gadobutrol MRI contrast agent	Bayer		Gadavist (Gadobutrol, 1mM/mL)
Stereotactic frame	Stoelting	#51500	not MRI compatible
turnkey FUS delivery device	FUS Instruments	RK-300	ready to use MRI compatible FUS for rodents