This article details a protocol for imaging brain structures in anesthetized rats using ultrasound and photoacoustic techniques. The methodology emphasizes the preparation of the animal and the precise alignment of imaging equipment to capture detailed anatomical and vascular information.
Position an anesthetized rat on the imaging station with a nose mask to maintain anesthesia.
Remove fur around the ears and neck to avoid interference with ultrasound imaging.
Arrange the animal to extend its limbs. Apply electrode cream to the sensors, and secure the paws to monitor vital parameters.
Apply ultrasound gel to the rat’s head and transducer for effective ultrasound transmission.
Then, incline the worktop to expose the temporal side of the head.
Align the transducer vertically to image deeper brain structures.
Set ultrasound B-Mode acquisition parameters. During imaging, the transducer emits ultrasound waves that reflect off tissue interfaces, generating a grayscale map of brain anatomy.
Adjust the transducer to align with the target brain region.
Switch to Color Doppler Mode to visualize brain blood vessels.
Adjust the parameters to enable real-time detection of blood flow velocity and direction.
Lay the rat down on an ultrasound and photoacoustic imaging station worktop, and quickly position the nose inside the appropriate mask, providing a constant anesthesia flow. To shave the animal, spread a consistent layer of hair removing cream on the head surface, covering areas surrounding the ears and neck. After allowing the cream to act for several minutes, gently take it out with a spatula. Softly remove all cream remnants with a wet sponge to accurately clean the skin.
To position the rat, arrange the animal in a spread eagle position. Lean the paws on the vital parameter sensors on the worktop after applying some drops of electrode cream to monitor the vital signs. Finally, fasten the limbs with a hypoallergenic artificial silk patch.
Then dispose a consistent layer of hypoallergenic, water-soluble ultrasound transmission gel on the animal's head. Cover the transducer head with a thin layer of the same gel, and put it into contact with the layer on the rat. Raise the animal head, and rotate it slightly on one side.
Use a cotton roll as a stand, keeping the snout well inserted into the anesthesia mask. Incline the worktop at an angle of about 30 degrees with respect to the horizontal plane. Turn the imaging transducer at an angle of about 30 degrees with respect to the vertical plane.
For ultrasonic and photoacoustic anatomic and vascular image acquisition, turn the imaging scan on. Enter the B-mode image acquisition, and properly set all image acquisition parameters to respect possible given requirements of the experiment. Set the transmit center frequency as low as possible in order to have the maximum penetration depth for the transducer.
Start image acquisition in B-mode, and adjust the transducer positioning in real time by identifying anatomical references and by centering the region of interest to the monitor middle point. Place the transducer to align it to the virtual axis, connecting the ear to the eye to obtain an optimal beam focalization. Acquire different views of the internal brain volume by clockwise or counterclockwise rotation. Ensure that the cerebral region of interest localizes at 10 millimeters of depth with respect to the ultrasound laser transducer source in order to receive an optimal photoacoustic response signal.
Next, enter color Doppler mode to visualize internal brain blood vessels in a highly sensitive way. Then, choose the desired acquisition parameter set in color Doppler mode. Acquire images in this modality to distinguish blood stream velocities and directions until several millimeters of penetration depth.
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