Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Our protocol provides a way to visualize the neuronal response to mechanical stress in a live mouse, which can provide direct evidence of how traumatic brain injury affects the brain. This technique can monitor the target protein at the same brain location in the same animal for both the acute and chronic phases after traumatic brain injury. The gene of interest and the virus promoter can be changed accordingly to study other proteins in the specific cell types in the brain.

To begin, apply ophthalmic ointment to the anesthetized mouse’s eyes. Remove the hair from the top of the head by trimming with regular scissors. After establishing a 12 to 15 millimeters long midline incision, excise the skin over the left and right hemispheres of the skull using curved tip spring scissors.

Once the skull is exposed, remove the periosteum by gently rubbing it with the sterile cotton tipped applicator, and flushing it with sterile saline. Once the skull area is dried, mark the Traumatic Brain Injury, or TBI, impact site at the coordinates 2.5 millimeters posterior to the bregma and two millimeters lateral from the sagittal suture to the right. Quickly remove the mouse from the stereotaxic frame and place the head on the buffer cushion under the TBI device.

Align the impactor tip with the marked impact site. Lift the metal column by pulling the tethered nylon string 15 centimeters above the mouse head, and then release it, allowing the weight to fall freely onto the transducer rod, which is in contact with the skull top at the TBI site. Place the anesthetized mouse’s head onto the stereotaxic frame kept under the surgical microscope.

Gently and slowly drill the surface of the skull using an FG4 carbide bit at a low rotor speed to remove the remaining periosteum, and create a rough skull surface such that the dental cement binds securely with the skull. Use saline to flush and clear bone dust from the skull surface. To separate the lateral muscles from the skull, at approximately five millimeters posterior to the eye, where the suture connecting the parietal and temporal skull is located, gently insert the fine closed tips and gently move the closed tips in the posterior direction until the lambdoid suture.

Separate the lateral muscles on the side where cranial window implantation will occur. Wash away the debris from the surgical site using saline and dry the area with gauze. To implant the head post, mark the center point 2.5 millimeters posterior to the bregma and 1.5 millimeters off the sagittal suture on the right hemisphere using a marker pen and a surgical caliper.

Then trace the circumference of the craniotomy on the clean and dry skull. Loosen the ear bar and rotate the head, so that the craniotomy plane is perfectly horizontal, and then tighten the ear bar again. Use the wood stick of a cotton tipped applicator to add two small drops of superglue to the front and back edge of the head post.

Position the titanium head post over the center of the craniotomy, and quickly adjust it to rest within the same plane where the cranial window will be implanted. Apply light pressure until the superglue is dried, which usually takes approximately 30 seconds. Prepare dental cement in a pre-cooled ceramic dish by thoroughly mixing 300 milligrams of cement powder, six drops of QuickBase liquid, and one drop of catalyst.

Quickly apply a generous amount of dental cement mixture to the outside perimeter of the traced circumference and cover any exposed bone surface. However, do not cover the site of the craniotomy. Release the ear bar and secure the head post to the metal frame to ensure that the head is stable for precise drilling along the marked craniotomy circumference.

To perform the craniotomy, use a surgical caliper to verify the diameter of the marked circle as demonstrated before, and adjust as necessary, so that the cranial window will fit snugly inside the craniotomy. Using an electric dental drill, etch and thin the skull along the outside of the marked circle using an FG4 carbide bit first, which creates a track within which to thin the skull. Irrigate the area with saline to wash away the bone dust.

Continue to thin the skull using an FG 1/4 carbide bit until the skull is paper thin and transparent. Periodically, stop drilling, and irrigate the whole area with sterile saline again to reduce heating from the drill and to wash away the bone dust. Complete the skull thinning using an EF4 carbide bit, and continue thinning and drilling through the rest of the skull along the track.

Insert a 0.5 millimeter fine-tipped forceps through the cracked place, and lift the bone flap gently upward without indenting the underlying brain. After removing the bone flap, irrigate the craniotomy area with saline. Using the curved tipped surgical forceps, gently remove the visible arachnoid matter.

Use the straight tipped surgical forceps to pick up the sterile glass window with the three millimeter glass cover slip facing down. Place and adjust the glass window above the craniotomy site to make sure that the window can fit snugly to the craniotomy edge. The five millimeter glass cover slip is on top.

Prepare the dental cement as demonstrated earlier, and wait approximately six minutes until the cement becomes pasty and thick. While waiting for the cement to become pasty and thick, apply an adequate amount of pressure to the window through a stereotaxic manipulator to check that the skull can securely and tightly contact the glass window. Use an adjustable precision applicator brush to add a small amount of cement alongside the window edge to seal the glass window with the skull.

Wait approximately 10 minutes to let the cement completely dry, then gently release, and remove the manipulator above the window. Complete by trimming the dental cement using the dental drill with an FG4 carbide bit if excess cement covers the window. At approximately four hours after surgery, two-photon imaging was carried out, where at the superficial level vasculature appeared black, and at the deep level of approximately 400 micrometers, EGFP protein expression was diffusely distributed throughout the cell body.

At one week and four months post TBI, the vasculature pattern observed at the day zero time point was used as a reference to locate the same imaging region. The vasculature pattern is similar to that at day zero. Similarly, the EGFP expression was detected throughout the cell body.

The fluorescence intensity at day zero and four months was similar at both the superficial and deeper levels. However, the fluorescence intensity at one week was lower than that of day zero and four months, possibly due to the translational repression reported for other TBI models. It is crucial to take utmost care when operating the craniotomy step to avoid damaging the brain tissue.

Do not get the drill tip inserted into the brain. Using this technique, researchers can visualize different proteins of interest in the same cell longitudinally across different phases post-TBI, providing information on the localization, expression, and solubility of that protein in the mammalian brain after trauma.