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

Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the blood-brain barrier. Normally the blood-brain barrier impedes the delivery of potential drugs for several brain diseases. Upon sonication, the injected microbubbles start oscillating and induce strain on the vessel wall, opening tight junctions and eventually allowing drugs to cross the blood-brain barrier In preclinical models, the effect of ultrasound and microbubble treatment are often studied by contrast-enhanced magnetic resonance imaging or by studying extravasation of dyes ex vivo.

The drawback of this approach is that the real time response during and immediate after ultrasound exposure cannot be acquired. Intravital multiphoton imaging of the brain gives the opportunity to study these effects in real time. In the following video, we will present stepwise instructions on how to place a cranial window in a mouse and perform ultrasound microbubble treatment using a ring transducer mounted to the skull.

This procedure enables concurrent sonication and in vivo multiphoton imaging. To begin prepare the materials needed for the cranial window surgery and ultrasound microbubble treatments. In this video, we show an acute non-recovery cranial window surgery.

For chronic recovery cranial window surgery, sterilized tools and materials, a sterile surgical space, and appropriate drugs are necessary. Prior to surgery administer anesthesia and analgesia. Remove the fur on the head.

Place the animal in a stereotaxic frame. Insert a catheter into the tail vein for dextran and microbubble injections. Maintain the animal's core temperature of 37 degrees by using a heat source.

Here we are using a glove filled with warm water. To remove the scalp, lift the skin between the eyes using forceps and cut along the sagittal suture. Remove the periosteum covering the outer surface of the skull with a cotton swab.

Afterwards mark the desired location of the cranial window on the skull and begin drilling along the outline. 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. Alternate between drilling and cooling the skull.

Check the drilling progress by applying gentle pressure onto the bone island. Remove the bone island by using a pair of forceps. Ensure that the brain is kept moist by using a piece of surgical sponge that has been presoaked in saline.

To place a cranial window, place a drop of saline onto one side of a coverslip and maneuver it over the hole in the skull. Spread a layer of glue around the perimeter of the coverslip to attach it to the skull. Once the glue is completely dry, deposit the agarose solution onto the window and place the transducer on top.

Apply firm pressure such that there's minimal agarose between the transducer and the cranial window. When the agarose has cooled to a Jell-O-like consistency, cut away the excess agarose from the circumferences of the transducer's coverslip. Spread a layer of glue along the circumferences of the transducer's coverslip to adhere it to the skull.

After the glue has completely dried, position the animal under the objective lens of the microscope. Ensure that the objective does not collide with the transducer or coverslip. Select a field of view in the multiphoton microscope.

Set up an imaging scan of the vasculature and begin sonication. For this protocol, we use a ring transducer that is operated at a driving frequency of 0.82 megahertz, 10-millisecond cycles mechanically indexed of 0.2 to 0.4, and pulse repetition frequencies between one and four Hertz. Sonovue microbubbles are injected at a dose of one milliliter per kilogram.

Successful ultrasound microbubble treatments can be detected by the extravasation of fluorescent dextran from the intravascular to the extravascular space, indicating an increase in BBB permeability. To evaluate kinetics of dextran leakage as a representative model for drug delivery, the signal intensity between the intra and extravascular spaces can be evaluated using tools such as MATLAB. To evaluate the vascular changes induced by ultrasound microbubble treatments, the diameter of the vessel of interest can be measured before, during, and after ultrasound microbubble treatment.

Image analysis can be conducted by using software such as ImageJ, FIJI, or programming tools. Here Imaris was used for blood vessel segmentation and classification. Intravital multiphoton microscopy of the brain combined with ring transducers provide a method to monitor the effects induced by ultrasound microbubble treatment in real time at a high spatial and temporal resolution.

Quantitative and qualitative data can be obtained to study, for example, extravasation kinetics and vascular changes. The experimental method presented here can be applied to several research models and to various ultrasound applications.