Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

All the following experimental procedures were approved by and conducted in accordance with the Norwegian Food and Safety Authority, Sunnybrook Research Institute's Animal Care Committee, and the Canadian Council on Animal Care.

1. Material preparation

1. Prepare the materials needed for the cranial window surgery and ultrasound-microbubble treatments. For chronic cranial windows, sterilized tools and materials, a sterile surgical space, and pre- and post-surgery drug administration are necessary^23,24,25.
2. Transducer and coverslip preparation
   1. Check the physical integrity of the transducer: look for cracks and dents. Ensure the electrodes on the top and side of the transducer are intact.
   2. Deposit cyanoacrylate glue into a small dish. Use an applicator to spread a thin layer of glue onto the surface of the transducer.
   3. Place the transducer onto the glass coverslip. Press down firmly for 20-30 s.
      NOTE: A 3D-printed mold can be used to facilitate the alignment of the glass coverslip with the ring transducer, ensuring firm and even pressure on the coverslip and ring transducer (Figure 2).
   4. Check for bubbles between the transducer and the coverslip. If there are bubbles, take the coverslip off and repeat from step 1.2.3., as air impedes ultrasound propagation. Cure overnight at room temperature.
   5. Once adhered to a glass coverslip, match the transducer (Figure 3).
      NOTE: This protocol uses an in-house manufactured lead zirconate titanate ring transducer (10 mm outer diameter, 1.4 mm thickness, 1.2 mm height)³⁵, matched to a 50 Ω impedance and 0° phase load with a custom matching circuit. The transducer is driven at 0.82 MHz in thickness mode, producing a circular focal spot approximately 1 mm beneath the coverslip. Ring transducers of similar properties (10 mm outer diameter, 1.5 mm thickness, 1.1 mm height) have been characterized²⁶ and used extensively for multiphoton microscopy experiments^{27,28,29,31,32,36}.
3. Transducer reuse and coverslip replacement
   1. Replace the coverslip if it is cracked or has debris (e.g., fur, glue) from the previous experiment. To remove the coverslip, dissolve the glue by submerging the transducer and coverslip in acetone for 20 min.
      NOTE: Acetone may affect the integrity of the transducer and/or electrodes. Check with the manufacturer before proceeding with this step.
   2. Check if the acetone has dissolved the glue by gently pulling on the coverslip with forceps. Check once every 10 min to avoid prolonged acetone exposure.

2. Animal preparation

1. Anesthetize the animal by using a mix of medical air, oxygen, and isoflurane in an induction chamber.
   NOTE: Use of oxygen as a carrier gas has been reported to affect the half-life of microbubbles^37,38 and diminish the magnitude of ultrasound-microbubble induced increases in BBB permeability²⁷, but may also reduce the risk of hypoxia and mortality³⁹. Choose carrier gases based on project aims and veterinarian advice. Injectable anesthetics such as a ketamine/xylazine cocktail may also be used; however, it is easier to control the plane of anesthesia and blood oxygen levels when using inhalable anesthetics.
2. Check that the animal has achieved a sufficient plane of anesthesia by performing a toe pinch. Weigh the animal to determine the dosage of dextran, microbubbles, and drugs to administer. Remove the fur from animal's head and place the animal onto a stereotactic frame.
3. For acute experiments, access to the systemic circulation must be established for dextran and microbubble injections. To achieve this, insert a 27 g catheter into a tail vein.
   NOTE: While retro-orbital injections are also possible, tail veins are recommended due to the limited working space in the head area during multiphoton imaging.
4. Transfer the animal onto the stereotactic frame and switch the anesthesia to the nose cone. Maintain the animal's core temperature of 37 °C using a heat source, such as a heating pad or a glove filled with warm water.
5. Monitor animal temperature by using a rectal probe, and animal physiology by using a pulse oximeter. Apply ophthalmic ointment. Inject appropriate pre-surgery analgesic and/or anti-inflammatory drugs (see Table of Materials).
6. Prior to starting the cranial window surgery, check the plane of anesthesia and the animal's heart rate, O₂ saturation, respiratory rate, and temperature.
7. To begin the cranial window surgery, remove the fur on the head by applying a depilatory cream and/or using fur clippers. Remove the fur from between the eyes to the anterior half of the neck (Figure 4A).
   NOTE: Prolonged contact with the depilatory cream will burn the skin. For chronic cranial window surgeries, wash the scalp with alternating wipes of betadine and 70% EtOH following fur removal. Prepare the surgical space for sterile surgery. Sterility must be maintained until Step 2.15.
8. To remove the scalp, lift the skin between the eyes using forceps held in the non-dominant hand, along the sagittal suture. Using curved scissors, remove the skin to expose the parietal bones (Figure 4B). Apply firm pressure with a cotton swab if there is bleeding from the skull or scalp; bleeding must be stopped before progressing to the next step.
   NOTE: For acute surgeries, the skin can be pushed back and adhered to the skull using liquid cyanoacrylate glue or tissue adhesive.
9. Remove the periosteum covering the outer surface of the skull using cotton swabs.
10. Using an operating microscope (6-25x) and a dental drill (0.5 mm drill burr, medium speed), outline a circle onto the parietal bone to mark the desired location of the cranial window on the skull (Figure 5). Avoid the sagittal suture, lambda, and bregma, as these areas are thinner and overlay large blood vessels.
    NOTE: To facilitate drilling, an outline of the cranial window can be drawn onto the skull using a marker and stencil (Figure 5A). For rats, it may be easier to drill a rectangular, instead of a circular, cranial window. Due to the thickness of the rat skull bone, use a 0.7 mm drill bit to outline the cranial window in the compact bone prior to using a 0.5 mm drill bit to complete the drilling process.
11. Apply gentle pressure with the drill bit; excessive pressure increases the risk of causing damage to brain tissue. To prevent the skull from overheating during drilling, drip saline onto the skull using a syringe, or apply a piece of surgical sponge soaked in saline.
12. Alternate between drilling and cooling the skull until the resulting bone island separates from the rest of the skull. Check the drilling progress by applying gentle pressure onto the bone island using forceps or the drill bit. Continue drilling until the bone island separates from the rest of the skull.
    NOTE: Small cracks in the thinnest areas of the skull are a good indication that drilling is almost complete. Attempting to remove the bone island prematurely can cause pieces of bone to penetrate into the brain tissue, damaging the dura and causing inflammation and bleeding.
13. Remove the bone island by using a pair of fine forceps to grasp the edges, or the upper compact bone layer, of the bone island (Figure 6A). Ensure that the brain is kept moist by applying a piece of surgical sponge that has been pre-soaked in saline. If bleeding is observed, place the surgical sponge on the region that is bleeding. Do not proceed to the next step until bleeding has ceased.
    NOTE: If bleeding persists after 5 min, the animal cannot be used for multiphoton imaging experiments. For rats, it may be necessary to remove the dura if it is thick. To remove the dura, use high magnification on the operating microscope and a pair of fine forceps.
14. To place a cranial window, pick up a glass coverslip with a pair of forceps, place a drop of saline onto one side, and maneuver it over the hole in the skull. Ensure that there are no air bubbles under the coverslip.
    NOTE: Use a 5 mm glass coverslip for mice, and 8 mm for rats. For rats, due to the thickness of the skull bone, use an agarose solution instead of saline to fill in the space between the coverslip and brain. The transducer and its coverslip can also be adhered directly onto the skull, instead of using a separate coverslip for the cranial window. For this option, proceed to step 3.1. Refer to Figure 1 for details.
15. Spread a layer of cyanoacrylate glue around the perimeter of the coverslip (Figure 6B) to attach it to the skull. Ensure that there is no glue under the coverslip. Apply pressure onto the coverslip to ensure that glue does not come into contact with the brain.
16. Once the glue is completely dry, even out the surface of the glue by using the dental drill. Ensure all glue debris is removed from the surgical area.
    ​NOTE: For chronic cranial windows, inject the necessary post-surgical drugs (see Table of Materials), provide ointment for wound care and soft foods, and recover the animal under a heat lamp.

3. Placement of the ring transducer

1. Prepare the 1% (w/v) agarose solution. In a small beaker or Erlenmeyer flask, add 0.1 g of agarose and 10 mL of PBS (1x) or saline. Boil the solution until the agarose has fully dissolved by placing the beaker onto a hotplate or heating the solution in a microwave oven (30-45 s).
2. Steps 3.2-3.5 are time-sensitive as the agarose solution cools quickly. Withdraw ~ 0.5 mL of agarose into a 1 mL syringe.
   NOTE: To protect the integrity of the brain, ensure that the temperature of the agarose approximates body temperature before use.
3. Deposit the agarose liberally onto the coverslip of the cranial window.
   NOTE: If the tissue blanches, the temperature of the agarose was too high; the animal must be euthanized. If there is no separate coverslip covering the brain (i.e., the transducer and its coverslip are placed directly onto the brain, see step 2.14), then agarose should be deposited onto the surface of the brain in this step.
4. Place the transducer over the cranial window (Figure 6C). Apply firm pressure such that there is minimal agarose between the transducer and the cranial window. Ensure that the transducer is centered (XY-plane) and parallel (Z-plane) to the cranial window, and that there are no air bubbles in the agarose.
5. When the agarose has cooled to a jello-like consistency, cut away excess agarose from the circumference of the transducer's coverslip using a spatula or scalpel. Ensure that there are no air bubbles underneath the transducer's coverslip.
6. Using a spatula, spread a layer of cyanoacrylate glue over the circumference of the transducer's coverslip, extending to the skull, such that the transducer is firmly adhered to the skull.
7. Maintain firm pressure on the transducer until the glue has completely dried (10-15 min).

4. Multiphoton microscopy imaging

1. Position the animal under the objective lens (Figure 7A). Ensure that the objective lens is centered in the ring transducer, and parallel with the transducer (Figure 7B). If a water-immersion objective lens is used, fill the center of the ring transducer with deionized and degassed water.
   NOTE: Degassed water is important for proper ultrasound propagation.
2. Start with the objective lens in its highest position, and then slowly lower the objective lens until it is within the ring transducer (Figure 7A, B). Ensure that objective lens does not collide with the transducer or coverslip.
   NOTE: Alternate between the eyepiece to check if the Z-position of the objective lens is in-plane with the surface of the brain, and by eye to ensure that the objective lens does not collide with the transducer or coverslip. It may be easier to visualize the pial vessels through the eyepiece following injection of fluorescent dextran through the tail vein (Figure 7C).
3. Prepare the multiphoton microscope for imaging.
   NOTE: This protocol uses an upright multiphoton microscope and a 20-25x objective lens that has a working distance of 2 mm, which is sufficient to focus beyond the coverslip(s), into the brain parenchyma.
4. Prepare the dextran. Add the appropriate amount of PBS to the vial of dextran, as per the manufacturer's instructions. Vortex the dextran solution for 1-3 min to ensure that the dextran powder is fully dissolved. Inject the dextran solution into the tail vein.
5. Setting up an image scan
   1. Using the eyepieces, ensure that the objective lens is parallel to the brain. Tilt the animal to correct for XZ and YZ misalignments.
   2. Select a field-of-view in a multiphoton microscope. Set up an XYZ scan before ultrasound exposure to have a baseline image of the vasculature prior to ultrasound exposure.
      ​NOTE: Typical imaging parameters are as follows: 300-800 µm in depth, 2-5 µm step-size, and 10-20 time stacks. Ensure that the objective lens does not come into contact with the transducer or coverslip at its lowest point during the imaging sequence.

5. Ultrasound exposure

1. Ensure all the BNC cables are connected correctly (Figure 3).
2. Set up an XYZT image scan that is sufficiently long enough to capture image stacks before, during, and after ultrasound-microbubble treatments.
3. Prepare the microbubbles by following the manufacturer's instructions. Inject the microbubbles into the tail vein and begin imaging.
   NOTE: Microbubble injections can be done with an infusion pump to ensure consistent injection rate and to enable concurrent microbubble injection and imaging. If microbubbles are to be injected during imaging, ensure that the tail vein can be easily accessed without exposing the detectors to ambient light.
4. Begin sonication.
   NOTE: Typical sonication parameters are as follows: 10 ms cycles, mechanical index of 0.2-0.4, and pulse repetition frequencies between 1-4 Hz. Sonication and microbubble parameters used in preclinical ultrasound-microbubble studies have been extensively studied and are well documented in literature (e.g. see ⁴⁰ for a review).
5. Continue multiphoton imaging throughout the duration of sonication and following the end of sonication. Be observant for dextran extravasation from blood vessels, as this is indicative of increases in BBB permeability.
   NOTE: If dextran is detected in the extravascular space, but in the periphery of the field-of-view, then there may be affected blood vessels outside of the field-of-view. This can result from misalignment of the transducer with the focus of the objective lens. In this scenario, it is easier to adjust the field-of-view by moving the objective lens or by repositioning the animal, than to realign the transducer.
6. Once imaging is completed, euthanize the animal cervical dislocation under deep anesthesia or CO₂ asphyxiation. For chronic cranial windows, spread a layer of dental cement onto the exposed skull.
   ​NOTE: For chronic cranial windows, the skin surrounding the window can be sutured, although this is not necessary, due to the removal of the scalp in step 2.8.

6. Image analysis

1. Export image stacks.
2. Analyze images with image analysis software (e.g., Olympus Fluoview, ImageJ/FIJI, Bitplane Imaris, ThermoFisher Scientific Amira) and/or programming tools (e.g., Python, MATLAB).