This method helps to answer whether blast traumatic brain injury results in cerebral vascular injury and if the scope of the effects extends to an impaired myogenic vascular response. The isolated pressurized arterial preparation allows us to separate the direct effects of blast exposure on cerebral arteries from the indirect effects caused by blast exposure to the brain. Isolated and pressurized arterial preparations can be used to study vascular function in a variety of diseases or conditions, including stroke, hypertension, and then diabetes.
The goal of our studies is to identify the therapies that maintain the ability of the cerebral circulation to protect the brain from the deleterious effects of hemorrhage. Demonstrating the procedures today will be Maggie Parsley, the assistant director of our laboratory and Yaping Zeng, our head research technician. To prepare the advanced blast simulator, or ABS, use a 2.54 centimeter piece of masking tape to tape along the top edge of four stacked pre-cut Mylar sheets.
Using a second piece of tape, secure the top edge of the Mylar membrane to the top of the expansion chamber over the center of the opening between the driver and expansion chambers. Replace the two all-thread rods at the top of the chamber and hand-tighten all of the cap nuts surrounding the chamber to secure the driver chamber against the Mylar membrane. Tighten the hydraulic hand pump knob and observe a constant pressure threshold on the hydraulic gauge to confirm that the driver chamber remains pressurized with no leaks.
Then open the trigger acquisition file that records ABS blast device pressure traces on the ABS blast device computer. To prepare the anesthetized rat for the injury, use long pick-up tweezers to position the tongue to the side and wipe along the inside of the throat with a lidocaine-soaked cotton swab tip before gently inserting a stylette containing an endotracheal tube into the trachea. Once intubated, insert the end of the ventilator hose to the outside end of the endotracheal tube and observe and confirm that the rat is breathing steadily and without difficulty.
Next, remove the ABS specimen tray from the specimen chamber and warm the tray under a heat lamp. While the tray is warming, shave the top of the scalp from above the eyes to between the ears and use scissors to cut a standard-sized foam ear plug from the base to the rounded tip into identical vertical halves. Then insert a halved piece into each ear tip, first along the ear canal, until contact is made with the tympanic membrane.
Removing the ventilator hose from the endotracheal tube, quickly but gently slide the rat into the top end of the specimen tray, carefully guiding the head through the head holder opening and rubber collar. When the rat is in place, reinsert the ventilator hose back into the endotracheal tube and verify that the rubber collar is secured firmly, but not tightly around the neck, and that the rat is laying in a lateral prone position. Then lock and secure the ABS specimen tray containing the anesthetized rat into the ABS specimen chamber and gently pinch the hind paw toes with long tweezers every three seconds until a withdrawal reflex response is elicited.
Click Start on the opened acquisition page on the ABS blast device computer and press and hold down the ABS blast device trigger in the acquisitioning window until the blast goes off, rupturing the Mylar membrane and administering the ABS blast injury. Right after the blast detonates, start a second timer to keep track of how much time elapses in minutes and seconds until the rat rights itself, post-injury, and place the rat in the supine position on the blue pad and warming for assessment of the rating reflex suppression. To extract the middle cerebral artery, use a number 10 scalpel blade to make a central one and a half inch vertical bone-deep incision from the top of the shaved scalp to the occipital condyle and use small bone rongeurs to open and separate the scalp skin from the skull bone.
Using large bone rongeurs, cut and extract the occipital, interparietal, and lower half of the frontal bones encasing the brain and use a surgical spatula to carefully excavate the brain from the skull once it has been freed of the surrounding bone. Deposit the harvested brain into a small glass Petri dish containing chilled physiological salt solution, or PSS, on a solid ice block under a dissecting microscope, and carefully remove both the left and right middle cerebral arteries, or MCAs, beginning at the circle of Willis and continuing laterally and dorsally for approximately four to five millimeters. Use micro forceps to gently clean the collected MCA segments and mount the arteries onto an arteriograph.
Cannulate the proximal end of each segment with a glass, 70 micrometer diameter micropipette and secure the pipettes with 10-O nylon sutures. Softly perfuse the lumina with PSS to remove any residual blood and cannulate the distal end of each segment with a second micropipette without stretching the arterial tissue. Secure the micropipettes with a second set of 10-O nylon sutures and place the arteriograph on the stage of an inverted microscope equipped with a camera and monitor.
Raise the reservoir bottles connected to the micropipettes to an appropriate height above the segments to equilibrate the MCA segments at a pressure of 50 millimeters of mercury for 60 minutes. At the end of the equilibration period, set the bottles at different heights to each other to examine the vessel dilatory responses, lower the reservoir bottles to reduce the intravascular pressure to 80 millimeters of mercury and allow the segments to equilibrate for 10 minutes. Then, reduce the intravascular pressure to 60, 40, and 20 millimeters of mercury, allowing the segments to equilibrate after each pressure change.
In this representative graph, the arterial diameter is depicted on the Y-axis as the percent change of the diameter at a baseline pressure of 100 millimeters of mercury. On the X-axis, the different intravascular pressures at which the arterial dilatory responses were tested are shown. For the sham injury group, the MCA diameters increased above baseline and continued to normally dilate as the intraluminal pressure was reduced from 100 to 20 millimeters of mercury.
In contrast, the MCA dilatory responses to the continuous imposed reduction in intravascular pressure in the observed 30 and 60 minute blast-induced traumatic brain injury groups were significantly reduced in their ability to dilate after blast exposure, indicating a significant impairment of cerebral vasodilatory responses to reduced pressure. The vessel extraction, recovery, mounting, and profusion techniques are tremendously intricate, and every effort of precise care should be taken when performing these procedures.
