Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

The overall goal of the following experiment is to image the neuronal activity of the mouse with manganese enhanced MRI. It is done by unilaterally stimulating the vibrancy and mapping the neuronal response in the contralateral barrel field cortex. The first step is to assemble and calibrate the ultrasound system consisting of an unfocused ultrasound transducer driven by the signal generator and amplifier for the experiment.

After an intraperitoneal injection of manganese, the blood-brain barrier is opened with ultrasound and microbubbles allowing the manganese to enter the brain parenchyma. Next, the mouse is subject to a neuronal stimulation paradigm, which results in higher accumulation of manganese in the more active regions of the brain. Finally, MRI images are obtained that show regional differences in T one weighted MR image intensity based on different levels of manganese accumulation within the brain.

In neuroscience, the mouse is the dominant model system for studying the genetic and molecular bases for brain development and disease. This technique provides a non-invasive tool for mapping neural activity in these mouse models. Not only can ultrasonic disruption of the blood-brain barrier be used for delivering manganese to the brain parenchyma for the purposes of functional neuro imaging, but it can also be used to deliver a wide variety of other compounds into the brain Parenchyma, including gadolinium based contrast agents or chemotherapeutics.

Generally, individuals new to this method will have difficulty calibrating the ultrasound system and handling the micro rubles. Demonstrating the procedure will be each one of the technicians in our laboratory. The ultrasound system consists of a single element ultrasound transducer with a diameter wide enough to cover the mouse brain and a center frequency in the range of two megahertz.

The transducer is driven by a signal generator and 50 decibel power amplifier, which produced the ultrasound pulse sequence. Calibrate the ultrasound system in a water tank. Place the hydrophone in the bottom of the tank and position the transducer over it with a three axis translation stage.

Then apply a short train of sinusoidal pulses to the transducer and adjust the stage to find the peak response, which should be at the center of the ultrasound beam and the transducer's natural focus. Next, make several acoustic pressure measurements over a range of input voltages and use a linear regression to estimate the relationship between input voltage and acoustic pressure. Find the pulse amplitude to generate the peak negative acoustic pressure of about 0.36 megapascals.

In this step, anesthetize the animal by delivering the isof fluorine through the nose cone. The nose cone apparatus should fix the animal's head precisely and reliably in the same position each time our device maintains the head in the skull Flat position during the experiment is a pneumatic pillow to monitor the respiratory rate and titrate the anesthetic to maintain a respiratory rate between 85 and 125 breaths per minute. At the same time, monitor the animal temperature with a rectal probe and maintain its body temperature using a heat lamp or blown air.

After that, insert a tail vein catheter and an intraperitoneal catheter to the animal. The next step is to trim its hair in order to demonstrate unilateral stimulation of the VII and map the neuronal response in the contralateral barrel field cortex. Cut the contralateral VII as close as possible to the skin surface without irritating the follicle or the surrounding skin.

Then transfer the animal to the ultrasound system and apply the ultrasound gel to the scalp lower thin plastic sheet containing a water column onto the head. Reach through the water column with a cotton tip swab to push out any air bubbles that get trapped in the ultrasound gel. Then position the ultrasound transducer in the water at its natural focal distance directly over the mouse brain and wipe the transducer with your finger to remove any trapped air bubbles.

Afterward, produce the perrin lipid microspheres by shaking the vial in the agitator for 45 seconds while working. Leave the violet room temperature, but store it in the refrigerator after the last use of the day prior to the first use. On subsequent days, agitate the stored vial in the agitator.Again.

Next, inject 0.5 millimole per kilogram of manganese chloride intraperitoneal, and wait for 10 minutes to allow its distribution. Then agitate the vial of microbubbles by hand for one minute to resuspend the microspheres. Subsequently withdraw 30 microliters plus the dead volume of the catheter.

Be careful not to inject air into the vial as this will degrade the remaining microbubbles. Immediately start the ultrasound pulse sequence and inject the microspheres through the tail vein catheter with minimum delay. Continue the in sonification for three minutes.

Then turn off the ultrasound system. After the manganese level in the brain has stabilized for about 40 minutes, turn off the isof fluorine and remove the nose cone. At this point, begin the neuronal stimulation paradigm to map the barrel field cortex.

Move a paintbrush in a circular motion at about one to five hertz through the contralateral vibra array for 90 minutes. Resume anesthesia after the stimulation, then continue to maintain the body temperature and titrate the isof fluorine level to a respiratory rate of 85 to 125 breaths per minute. Next, place the mouse in an MRI coil and transfer it to the MRI system.

Then acquire the high resolution three DT one weighted images as shown here. Disruption of the blood-brain barrier after the administration of a T one shortening contrast agent such as manganese or a gadolinium based agent, results in a signal increase in the brain parenchyma on T one weighted imaging when compared to the brains in which BOMA was not performed. Here are the neuronal activity maps generated by unilaterally stimulating the risi in seven animals in the first column.

Difference maps at two different locations show increased MRI signal contralateral to the stimulated by brissy. In the second column, p-value maps indicate the relative significance of these differences. In the third column, the corresponding brain atlas diagrams show that these active regions correspond to the barrel field of the primary sensory cortex.

With practice, this technique can be performed in about two hours plus the duration simulation. When performing the procedure, it's important to remember to agitate the vial of microbubbles prior to injection and to be careful not to inject any air into the vial. In addition, it's important to make sure no bubbles of air are trapped in the ultrasound gel or underneath the ultrasound transducer.

For this demonstration, we've chosen to stimulate the whiskers, but clearly this technique can be adapted to study any stimulus of interest. Following the procedure, the animal can recover completely allowing longitudinal studies.