Two-Dimensional Super-Resolution Visualization of Rat Brain Microvasculature Using Ultrasound Localization Microscopy

The scope of our research focuses on developing a protocol for ultrasound localization microscopy to achieve super resolution imaging of the rat brain microvasculature. We aim to address the challenges of balancing special resolution and the penetration depths in imaging small vessels, providing detailed insights into vascular structure and blood flow dynamics in healthy and the disease states.

Our protocol offers the advantage of achieving a high spatial resolution of 12.5 micrometer, while maintaining a penetration depths of up to 12 millimeter, surpassing conventional methods, like MRI, CT, and double ultrasound. Unlike optical techniques, it enables imaging deep brain regions, reconstructing microvascular structures, and visualizing blood flow dynamics simultaneously.

Our results paves a way for investigating microvascular changes in neurological disorders, such as glioblast tumor and the Alzheimer's disease. The ability to visualize blood flow dynamics and the microvascular architecture with high resolution opens new opportunities to study disease progression, treatment efficacy, and the relationship between muscular alterations and the brain function.

[Narrator] To begin, position the anesthetized rat on the operating table. Secure the rat's upper incisors in the notch of the incisor bar, positioning the lower jaw beneath the bar. Position the ear bars in the bony indentation, slightly anterior and superior to the ear canal, ensuring the cranial surface remains horizontal. Apply a small amount of pressure to different parts of the head to assess stability. Fine tune the height of the imaging platform and adjust the angle of the rat's head using the incisor bar notch to ensure unobstructed breathing. Apply erythromycin ointment or 30% glycerin solution to the rat's eyes to protect them from surgical lights and maintain moisture. With a handheld electric clipper, shave the rat's head against the direction of hair growth, covering the area between the ears and from the eyes to the neck. Now, make an incision along the sagittal suture of the rat's skull, starting just the occipital bone and extending approximately four centimeters anteriorly. Use hemostats to retract the skin on both sides. Optionally, excise the skin over the skull for greater access. Use small scissors to remove the periosteum from the skull, fully exposing the hard bone layer. Perform the craniotomy with a handheld mini cranial drill, having a 2.5 millimeter spherical drill bit. Gently tap to abraid the bone, drilling for two to three seconds at a time, starting centrally and progressing outward. Inject approximately one milliliter of 0.9% sodium chloride solution every two minutes into the drilling area to cool and rinse away debris. Switch to a finer one millimeter drill bit once the white bone tissue no longer appears consistently connected. Continued drilling until central major blood vessels are clearly visible as dark brown with the surrounding tissue appearing pink and micro vessels as slightly reddish. To prepare the contrast agent, dissolve SF6 gas and lyophilized contrast agent powder in five milliliters of 0.9% Sodium chloride. Vigorously shake the mixture to form a microbubble, or MB suspension, with a final SF6 concentration of eight microliters per milliliter. Draw 0.8 milliliters of the MB suspension into a one milliliter syringe attached to a microinjection pump. Insert a 26 gauge in-dwelling needle with a catheter into the rat's tail vein and inject. Before imaging, mount a probe with a central frequency of 15.625 megahertz on the manipulator arm of the brain stereotaxic instrument equipped with a clamp. Position the ultrasound probe directly above the exposed rat brain, and apply coupling gel to the exposed brain surface to ensure optimal signal transmission. Open the software's main interface, which integrates a wrap brain atlas with motor motion control programs. Set the BGMA point as the origin, and use the software's real-time display to monitor the probe's trajectory and the corresponding rat brain slice location. Select the target imaging plane, such as BGMA minus one millimeter. Launch MATLAB 2021a software, and enter the data acquisition script in MATLAB 2021a. In the root directory, type "activate" in the command line window to activate the runtime environment. Then set the data collection start and end depths at five and 120 wavelengths, respectively, to effectively capture the region of interest. Set the plane wave transmission steering angles from minus five degrees to five degrees in 2.5 degree increments to enhance image resolution and contrast. The ultrasound localization microscopy revealed clear visualization of microvessels at depths up to 12 millimeters. Analysis of full width at half maximum indicated the smallest detectable vessel diameter as 13 micrometers. Fourier ring correlation confirmed the spatial resolution of microvascular imaging as 12.5 micrometers. The blood flow directions in a cross-sectional slice of the rat brain demonstrated downward flow in small cortical arteries and upward flow in small veins, represented by blue and red colors, respectively. A velocity map showed higher flow rates in larger vessels, with the majority of velocities concentrated in the 10 to 25 millimeter per second range. Imaging of the glioblastoma rat model showed abnormal vascular dilation, structural irregularities near the tumor, altered blood flow patterns in the tumor region, and a heterogeneous vascular flow within and surrounding the tumor.