Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

All animal studies were approved and conducted in accordance with the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC RA20M223). Only adult Sprague-Dawley female rats (average weight: 250 g; age: 11 weeks) were used for the present study.

1. Low-intensity focused ultrasound assembly and setup

1. Acquire a focused ultrasound transducer system with specifications sufficient to achieve BSCB opening in rats. Suggested parameters from the literature include a central frequency between 0.25-4 MHz and the capability of producing peak pressures between 0.2-2.1 MPa ^{10,14,15,16,17}. Ensure that the system includes the driving/control equipment, which at a minimum includes a wave/signal generator, radio-frequency (RF) drive/power amplifier, and matching network (Figure 2A).
   NOTE: The setup described here uses a commercially available multi-element transducer with a central frequency of 250 kHz and 64 mm diameter (Figure 2B).
2. Affix the 3D-printed probe holder and water cone on the transducer (Figure 2C). Ensure a watertight seal between the cone and transducer.
   NOTE: A custom cone and probe holder came included with the transducer used in this Experiment. The cone and probe holder affix to the transducer with screws, which are also provided.
3. Sterilize a 50 µm thick, acoustically transparent polyester membrane and affix to the bottom of the water cone using a rubber band.
4. Fill the water cone with degassed and deionized water using the inlet and outlet tubes. Take care to avoid air bubbles inside the cone, as they can disrupt acoustic coupling between the transducer and target. The polyester membrane should be slightly inflated.
   NOTE: To remove air bubbles from the cone, guide the bubbles to the outlet valve while filling the cone with water through the inlet valve. If many small bubbles are present, close all the valves and rotate the cone until one big bubble remains. Guide this bubble to the outlet valve and resume filling the cone.
5. Connect the driving equipment, which includes the wave generator and RF drive amplifier, to the transducer. The transducer cable will connect to the output side of the matching network, and the signal generator/power amplifier will connect to the input side of the matching network. The cables should be connected to their corresponding channel number (Figure 2D-G).
   NOTE: In the commercial system used in this study, the wave generator and RF drive amplifier are components of the transducer power output (TPO) (Figure 2D).
6. Attach the probe holder to the stereotactic arm. Affix the stereotactic arm to the fixation plate assembly. This will allow the transducer to be positioned precisely above the rodent during sonication.

2. Animal preparation and surgical laminectomy

1. Anesthetize the rat with a mixture of isoflurane and medical air in an induction chamber attached to a charcoal filter canister. Set the gas flow rate to 400 mL/min and the isoflurane vaporizer between 1.5%-2.5% for anesthesia induction. The amount of time spent in the chamber before complete sedation is variable, though it typically ranges from 3-6 min.
2. Record the weight of the sedated rat and perform a toe pinch test. If jerking or movement is observed in response to the pinch, place the rat back inside the induction chamber for an additional 1 min and repeat the toe pinch test. Repeat as necessary to ensure the rat is and remains fully anesthetized.
3. Place a heating pad and sterile absorbent pad on the fixation plate. Position the rat on the absorbent pad, apply eye ointment, and place a rectal thermometer to monitor body temperature.
   NOTE: During the length of the surgical procedure, the rat's temperature and heart rate should be monitored (ideally, the heart rate should be between 330-480 bpm and the temperature between 35.9-37.5 °C)^18,19. Adjust the isoflurane or heating pad accordingly to prevent premature death. The heating pad can be set to a temperature around 37 °C and should be turned on and off as needed to maintain the optimal body temperature.
4. Palpate the last rib of the rat, which is attached to the spine at the 13^th thoracic vertebra (T13). Use an electric razor to shave the fur off the dorsal surface between the last rib and neck. Wipe the exposed skin with gauze dipped in 10% iodopovidone.
5. Create a midline incision using iris scissors and dissect through the fascia until the spinous processes and lamina are exposed. Remove bone with offset bone nippers and angled blade iris scissors until the spinal cord is exposed²⁰. The length of laminectomy and incision varies based on the number of different targets to be sonicated. In this study, a three-level laminectomy was performed using a 3 cm incision.
   NOTE: Avoid touching or placing pressure on the spinal cord while removing bone to prevent injury. If the rat's hindlimbs jerk during the laminectomy, then too much force was used on the cord or nerve roots.
6. Secure the rat to the fixation plate by clamping the spinous processes adjacent to the laminectomy. Slightly pull the spine taut to minimize curvature before locking the clamps.

3. Target localization using laser guidance

1. Adjust the position of the transducer with the stereotactic arm until it is located exactly above the laminectomy (Figure 3A). The frame allows movement in the x-, y-, and z-axes, as well as 180° rotation in the vertical plane and 360° rotation in the horizontal plane.
2. Affix the laser apparatus to the bottom of the water cone and lower it until the laser point is visible. Adjust the lateral position of the transducer until the laser point is above the location that is the target for BSCB disruption (Figure 3B,C).
   NOTE: A computer-aided design (CAD) file for the laser apparatus is included in the supplemental section (Supplemental Figure 1).
3. Remove the laser apparatus and fill the space between the cone and spinal cord with degassed ultrasound gel (Figure 3D). For maximal coupling, ensure that no air bubbles are present in the gel.
   NOTE: In this study, the transducer with an affixed water cone was lowered until located 1 cm above the cord. Since the water cone was 30 mm in length, the total distance from the transducer to the cord was 40 mm. The water cone was placed 1 cm away from the spinal cord because the rat's skin, fascia, and musculature on either side of the incision prevent direct contact between the tip of the cone and the cord. Using the numbers on the stereotactic arm's y-axis may be helpful in keeping track of the vertical distance at which the cone is 1 cm away from the cord, especially since the gel will make visual confirmation of the cone's distance from the cord difficult.
4. Set the parameters for sonication on the TPO. A range of values can be used to achieve successful BSCB disruption. For maximal power, set the sonication frequency close to the center frequency of the transducer. The values used in this study are listed in Table 1.
   NOTE: The parameters listed here were adapted from prior work with LIFU, with a center frequency of 500 kHz, tone burst duration of 500 µs, a duty cycle of 50%, and sonication times of 5 or 10 min to safely neuromodulate a rodent spinal cord²¹. Based on studies that successfully achieved BSCB disruption, other parameters that can be used are central frequencies between 500 kHz-1 MHz, pressures of 0.2-2.1 MPa, burst lengths of 10-25 ms, and sonication times of 2-5 min^6,10,11,22.  

Table 1: Sonication parameters used for BSCB disruption.

4. Microbubble administration

1. Prepare a MB solution in accordance with the instructions provided by the manufacturer. Avoid introducing air into the solution.
   NOTE: The MBs are fragile and clump together near the top of the vial/syringe if left still for a few minutes. Shake the vial and syringe regularly to prevent an uneven dispersion of MBs. MBs have short lifespans; check the manufacturer's guide to determine the expiration time.
2. Insert a 22 G tail vein catheter and flush with 0.2 mL of heparinized saline (500 IU/mL)²³. To increase the chances of successful tail vein catheterization, dip the tail in warm water and place a tourniquet at the base of the tail to enlarge the vein's diameter.
   NOTE: Tail vein catheterization can be conducted prior to animal laminectomy, positioning, and targeting to save study time.
3. Inject 1 mL/kg of 3% EBD into the catheter. Flush with 0.2 mL of heparinized saline. The rat's extremities and eyes will turn blue. Confirm successful tail vein catheterization by checking for blue color change in the dorsal spinal vein of the rat (Figure 4).
   NOTE: EBD can be injected well prior to MB injection and will not influence sonication. In addition, since the Food and Drug administration (FDA) currently has not approved sonication with drugs already in the system, EBD can also be administered after sonication. This will result in less dye uptake, but may be more clinically relevant.
4. Inject a 0.2 mL bolus of MBs into the catheter and flush with 0.2 mL of heparinized saline. Start the sonication 1-2 min after the injection of MBs. The setup used here does not collect real-time sonication feedback.
   ​NOTE: Studies for BSCB disruption typically use a higher concentration of MBs than indicated for diagnostic imaging. Some concentrations of common MB brands used for BBB and BSCB disruption in rat models include 0.02-0.2 mL/kg and 200 µL boluses^10,15,24,25.

5. Spinal cord extraction and tissue processing

1. After completion of sonication, transcardially perfuse the rat with 100 mL of cold phosphate-buffered saline (PBS) until the blood runs completely clear. The liver, which is a rich blue color due to the dye, should fade to a light brownish-blue²⁶.
   NOTE: The purpose of perfusion is to remove excess blood from the vasculature of the spinal cord. Since EBD binds to albumin, this also removes excess EBD. This ensures that any EBD detected either visually or through fluorescence microscopy in the spinal cord is from the extravasation of dye into the spinal parenchyma.
2. Transcardially perfuse with 100 mL of cold 4% paraformaldehyde (PFA). The rat's limbs will twitch during this fixation if done thoroughly. This perfusion with PFA euthanizes the rat.
3. Remove the spinal cord and place it in 4% PFA at 4 °C overnight. Replace the PFA with PBS the following day.

6. Visualization of BSCB disruption

1. Isolate a 2 cm section surrounding the location of sonication using a razor blade. Split the section down the midline with the blade and section into 10 µm thick sections using a microtome. For bright-field visualization, stain with hematoxylin-eosin (H&E) stain.
   NOTE: The H&E spinal cord samples shown in this study were stained with hematoxylin for 3 min and eosin for 1 min²⁷.
2. For fluorescence microscopy, deparaffinize the slides containing the spinal cord sections and counterstain with 25 µL of 4′,6-diamidino-2-phenylindole (DAPI) dissolved in the mounting medium (0.5 µg/mL). Incubate at 4 °C for at least 10 min. Avoid light to prevent bleaching.
   NOTE: The deparaffinization can be replaced by using a cryostat to obtain frozen sections.
3. Use a fluorescent microscope to image all the slides. EBD autofluorescence (excitation: 470 nm and 540 nm; emission: 680 nm) is visible in the red channel, while DAPI is present in the blue channel. Use a light microscope to image the H&E slides.
   NOTE: Although this protocol described a non-survival procedure, it was also performed using survival surgical techniques. For survival surgery, disinfect the skin before incision with 3 alternating applications of iodopovidone and administer buprenorphine subcutaneously (0.05 mg/kg) before surgery. Continue to provide subcutaneous buprenorphine every 12 h at least 3 days post-operatively, with additional days if the rat exhibits signs of pain. If spinal cord injury occurs, rats may exhibit urinary retention or abnormal gait. This will present as dragging or delayed movement of the hind limbs or palpable, distended bladders. If this occurs, house rats with nutritionally fortified water gel for food and hydration and manually express bladders twice per day until reflex voiding is recovered. If there is complete paralysis of hind limbs or intractable pain, euthanize the rat.