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

Summary

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.

Abstract

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.

Introduction

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's disease, amyotrophic lateral sclerosis, and Alzheimer'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 agents^3,4,5.

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.

Protocol

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:

1. 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.
2. Remove the rats from the chamber and shave hair from the anterior and posterior areas of the neck using a hair removal (depilatory) cream.
3. Apply lubricant ointment to the eyes.
4. 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).
5. 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).

2. Stereotactic-guided focused ultrasound system

NOTE: The animals were transferred and placed on a table in a stereotactic FUS system. The rat'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:

1. 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.
2. Place ultrasound gel on the rat's head, water coupling bag, and the transducer (1 MHz frequency). Ensure that no air bubbles are present between the rat's skin, the water coupling bag, and the transducer.
3. Connect a coaxial BNC cable of the transducer to the input of the FUS system module.
4. In a control computer) Click on load for pre-registered rat images; a number of focal spots and acoustic pressure are chosen.
5. 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.
6. Set up the sonication parameters described in step 3.

3. Microbubble infusion and sonication

1. Activate the microbubble.
   NOTE: The microbubble used in this study was characterized by a diameter of 1.7 µm and a concentration of 0.2 × 10⁹ particles per milliliter¹⁰. The microbubble vial was activated based on the prescribing instructions provided by the vendor and a previously published report¹¹.
   1. Prepare a 5 mL syringe with a luer lock tip and filled with 5 mL of additive-free 0.9% sodium chloride injection.
   2. Remove the green cap, and connect the syringe to the vented dispensing pin (see Table of Materials) by screwing it clockwise.
   3. Remove the pin protection and place it in the center of the vial's rubber stopper. Press down firmly until the spike is fully inserted into the stopper.
   4. Empty the entire 5 mL syringe into the vial by pushing on the plunger rod.
   5. 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.
   6. To obtain the required dose, invert the syringe, and slowly withdraw the intended volume of the suspension into a 5 mL syringe.
2. 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.
3. 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) 'Burst mode' in the sonication mode menu in the FUS system; (7) Sonication time: 120 s.

4. MRI procedure using a 3T preclinical scanner

1. After sonication, transfer the animals to a table in a preclinical 3T cryogen-free scanner.
2. 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).
3. 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.
4. 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 µL of the MRI contrast agent and followed it with a 200 µL saline solution to flush the tubing.

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:

1. Perform a thoracotomy using a surgery scissor to expose organs in the chest and visualize the cardiac region.
2. Incise (~5 mm) the right atrium.
3. Introduce saline solution into the left ventricle for perfusion purposes.
4. 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.

Results

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...

Discussion

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...

Materials

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