The blood-spinal cord barrier is tightly bond and has limited permeability. This protocol allows researchers to safely and transiently open the blood-spinal cord barrier, using microbubbles and focused ultrasound. This technique allows blood-spinal cord barrier opening to be localized to the targeted spinal cord segment.
Additionally, the disruption can be easily confirmed via visual observation or fluorescent microscopy. This protocol can be explored for its potential in improving the delivery of gene therapies, or drugs to the spinal cord for the treatment of tumors, atrophy or injury. Demonstrating the procedure will be Meghana Bhimreddy.
A stellar MD student from my laboratory, who on a daily basis bridges the gap between engineering and medicine. To begin, acquire a focused ultrasound transducer system with specifications sufficient to achieve blood-spinal cord barrier, or BSCB openings in rats. Affix the 3D printed probe holder and water cone on the transducer.
Ensure there is a watertight seal between the cone and transducer. Fix the sterilized, 50 micron thick, acoustically transparent, polyester membrane to the bottom of the water cone using a rubber band. Fill the water cone with degassed and deionized water using the inlet and outlet tubes.
Avoid air bubbles inside the cone, as they can disrupt acoustic coupling between the transducer and target. At this stage, the Mylar membrane should be slightly inflated. Connect the driving equipment containing the wave generator and radio frequency drive amplifier to the transducer.
Fix the stereotactic arm to the fixation plate and attach the probe holder to the arm. Record the weight of an anesthetized Sprague-Dawley female rat, and perform a toe and tail pinch test, to confirm the anesthesia. 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. Palpate the last rib of the rat, which is attached to the spine at the 13th thoracic vertebrae.
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. Create a midline incision using iris scissors and dissect through the fascia until the spinous processes and lamina are exposed.
Remove the bone with offset bone nippers and angled blade iris scissors, until the spinal cord is exposed. Secure the rat to the fixation plate by clamping the spinous processes adjacent to the laminectomy. Then slightly pull on the clamps to make the spine taut.
Adjust the position of the transducer with the stereotactic arm until it is located exactly above the laminectomy. Affix the laser apparatus to the bottom of the water cone and lower it until the laser point is visible. Then adjust the lateral position of the transducer until the laser point is above the target location for BSCB disruption.
Remove the laser apparatus and fill the space between the cone and spinal cord with degassed ultrasound gel, without introducing air bubbles. Set the parameters for sonication on the transducer power output. Prepare a microbubble solution according to the manufacturer's instructions.
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 veins diameter. Then insert a 22 gauge tail vein catheter and flush with 0.2 milliliters of heparinized saline. Inject one milliliter per kilogram of 3%Evans Blue Dye, or EBD into the catheter.
Then flush the catheter with 0.2 milliliters of heparinized saline. Confirm the successful tail vein catheterization by checking for blue color change in the skin, eyes, or dorsal spinal vein of the rat. Inject 0.2 milliliters bolus of microbubbles into the catheter and flush with heparinized saline before starting the sonication.
After euthanizing the rat, remove the spinal cord and place it in 4%paraformaldehyde at four degrees Celsius overnight. The following day replace the paraformaldehyde with PBS. To visualize BSCB disruption, isolate a two centimeter section surrounding the location of sonication, using a razor blade.
Split the section into 10 micron thick sections using a microtome and stain with hematoxylin eosin stain. For fluorescence microscopy, deparaffinize the slide containing the spinal cord section, and counterstain with 25 microliters of DAPI dissolved in the mounting medium. Incubate the sections at four degrees celsius for 10 minutes in the dark to prevent bleaching.
After incubation, use a fluorescent microscope to image all the slides. Image the hematoxylin and eosin stained slide using a light microscope. Spinal cord vasculature is visible after laminectomy, and shows the posterior spinal vein, with multiple smaller vessels radiating laterally.
After intravenous injection of EBD, the surrounding tissue and spinal cord vasculature appeared blue. After sonication, a blue spot becomes visible over the targeted location, indicating the extravasation of EBD into the white parenchyma, due to BSCB disruption. Excised spinal cord from the rats with low intensity focused ultrasound, or LIFU sonication, confirmed apparent extravasation of EBD into the spinal cord.
While negative controlled rats without LIFU treatment did not show EBD extravasation. Spinal cords with LIFU sonication showed significantly greater intensity of EBD autofluorescence, than cords that did not receive sonication. With similar intensities of DAPI present in both.
Hematoxylin and eosin analysis revealed no neuronal damage, hemorrhage, or cavity lesions in the sonicated locations. Examples of injured cords due to surgical mishandling, and high powered sonication are shown as a comparison. In rats that received microbubbles and LIFU treatment, no change in motor scores, pre-sonication, post-sonication, and during a five day survival period was observed.
Minimal changes in spinal cord temperature were observed before, during and after sonication analysis. It is important to limit surgical damage to the cord during the laminectomy. Visualizing the H and E stained sections with microscopy, will indicate if any damage has occurred.
Following blood-spinal cord barrier disruption, using focus ultrasound, animals can be injected with antineoplastic agents, or gene therapies. The outcome will determine if this technique can improve delivery of therapeutics, and extend survival in the setting of spinal cord pathology.
