The overall goal of this procedure is to induce a mild to moderate brain injury in mice to model human traumatic brain injury. This is accomplished by first performing a C craniectomy. The second step of the procedure is to attach a hub to the skull over the craniectomy site.
Next, a pulse of water is delivered through the hub to induce brain injury. The final step of the procedure is to remove the hub and close the wound. Ultimately, results can be obtained that show morphological and behavioral changes at various intervals post-injury through immunofluorescence, microscopy, cognitive and motor testing.
Generally, individuals new to this method will struggle because it is difficult to perform the craniectomy without causing a breach in the dura, and also to attach the hub without getting glue on the dura. Therefore, visual demonstration of this technique is extremely valuable. Demonstrating the procedure will be fuka A technician from my laboratory Begin by preparing the surgical area.
Equip a stereotaxic instrument with a gas head holder for continuous inhalation of anesthesia during surgery. Next position a surgical microscope and light source as needed. Prepare the warming pad that will go under the mouse during surgery and ensure that all tools and materials that contact the surgical site are sterile.
Once the surgical area is ready, make the hub that will attach to the skull and connect to the LFP device. Use a razor blade to cut off the metal end of a 20 gauge one and a half inch needle cutting with a slight bias. The hub should have an opening of approximately three millimeters meters.
Next, cut around one millimeter thick disks from weed whacker line. These will be used to stabilize the tine while drilling the skull. After the mouse is anesthetized, place the animal onto the stereotaxic instrument and secure the head within the nose cone.
Remember to visually monitor the breathing rate during the surgery. Next, apply ointment to the eyes to prevent drying and remove the hair from the head. Clean and sterilize the surgical site with repeated applications of povidone iodine solution, followed by 70%alcohol.
Now that the surgical site is prepared, use a scalpel to make a midline incision starting from the eyes and extending to the neck. Pull back the skin with small bulldog clamps. To expose the skull and provide a clear surgical field, apply the topical anesthetic bupivacaine to the skull using a dental tool or bone scraper, remove the facia from the top of the skull.
Next, make a mark halfway between Bgma and Lambda and between the sagittal suture and lateral ridge over the right hemisphere. Place a drop of Loctite glue onto a plastic webo and use side grasping forceps to dip one of the discs prepared earlier into the glue. Press the disc to the mark made on the skull.
Use a needle and syringe to add one to two drops of the Loctite Accelerator. To harden the glue, place a three millimeter outer diameter triffin over the disc and spin clockwise until you have cut through the skull.Check. The tray finds progress frequently to avoid drilling too far and breaching the dura.
The skull flap should feel loose When pressed lightly. Place a dental tool parallel to the skull's surface and use it to pry up the bone flap. Carefully detach the bone from the skull with forceps.
Apply pressure with a cotton applicator to stop any bleeding and prevent bone dust from getting onto the dura. If there is a visible breach in the dura, the animal should be eliminated from the experiment by humane euthanasia. Use forceps to place the hub in a position over the hole such that the bias of the hub aligns with the curvature of the skull.
Use a sharp tipped wooden stick to apply super glue around the edges of the hub with one hand while stabilizing it with the other hand. Take care not to get glue on top of the dura as this may cause reduction in the force of the injury or induce a rip when the hub is later removed. Mix together Sano acrylic and dental acrylic in a small paper cup.
Use a one cc syringe to apply cement around the hub. The cement should cover the bottom two millimeters of the hub as well as the surrounding exposed skull. Fill the hub with sterile saline to keep the durra moist during the recovery phase.
A leak is likely if the solution fails to stay level and a new hub should be attached to finish administer fluids to the animal and remove it to a warmed empty cage with no wire lid. Place some hydrogel in the bottom and allow the mice to recover for one to two hours. The components of the LFP device are a pendulum, a water-filled acrylic cylinder with high pressure tubing and a male lure lock fitting attached, an amplifier and an oscilloscope.
To begin turn on the oscilloscope and amplifier. Confirm that the LFP device and the high pressure tubing connected to it are filled with water and free of air bubbles with the male end of the lure lock tube.Enclosed. Release the pendulum to deliver a test pulse.
Deliver several pulses and confirm that the pendulum gives a smooth signal on the oscilloscope and amplifier. A noisy signal indicates air in the system that must be removed prior to delivering the injury Pulse. Monitor the pressure delivered by the device to maintain the consistency of injury.
The oscilloscope trace here shows a peak-to-peak value of 2.16 volts, indicating a pressure of 1.47 atmospheres. The angle of the pendulum can be increased or decreased to modify the intensity of the pulse. Here, the angle is approximately 10 degrees.
After adjusting the LFP device, be sure to open the lure lock end of the tubing. Once the LFP device is ready, place on anesthetized mouse on the platform and fill the hub with sterile saline. Attach the male lure lock from the LFP device to the female lure lock fitting of the hub.
Place the animal on its side and once a normal breathing pattern resumes, but prior to the animal regaining sensitivity, release the pendulum to cause a single pulse of injury. The exact pressure of the pulse should be recorded. SHA animals undergo all of the same procedures with the exception of the actual fluid pulse to induce injury.
Place the animal on its back to monitor the writing reflex time. After the animal writes itself, it is again briefly anesthetized. Remove the cement and hub from the skull.
Note the presence of any herniation of the dura, and then close the wound with vet bond tissue adhesive. Visually inspect the hub for any occlusions. Place the animal into a warm cage until ambulatory and then return it to its home cage.
Behavioral testing following LFP can be used to assess both cognitive and motor outcomes. The Morris Water Maze is used to determine effects on learning and memory using visual cues in the testing room. Sham mice become more efficient at locating the platform.
With each subsequent day of training, my subjected to LFP take longer to locate the hidden platform on the first two days of testing relative to sham mice, but then appear to learn the task the third day. To determine the effect of injury on memory retention. A probe trial is performed one day after the last training session.
Sham mice spend more time in the target quadrant compared to the mice subjected to LFP. The motor deficits caused by LFP can be determined using the rotor rug test M subjected to LFP have a shorter average latency to fall compared to the sham mice at one seven and 21 days post-injury. Ggl fibrillary acidic protein staining reveals increased gliosis throughout the cortex in the region of injury, whereas sham mice do not display increased astrocyte numbers in the equivalent site.
Similarly, MAC one staining demonstrates more macrophages surrounding the site of injury compared to mice subjected to sham surgery. In addition, there is frequently physical damage to the cortical tissue visible in mice subjected to LFP, but not in sham mice. While attempting this procedure, it is important to remember that you should not breach the dura and that the LFP device should be clear of air bubbles.
This technique is allowing neuroscientists to evaluate various pharmacological and genetic manipulations before or after injury. In mice. This will help reveal what molecules reduce neural degeneration or promote regeneration in patients suffering from traumatic brain injury.
After watching this video, you should have a good understanding of how to induce mild to moderate traumatic brain injury in a rodent model by performing craniectomy, attaching a hub to the skull, and sending a pulse of water pressure onto the dura matter through the hub.
