A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

This model can be utilized to study the pathologies associated with the cerebral contusion, including intracranial hemorrhage, iron toxicity, neuron death, axonal injury, neurologic deficit, and the neural inflammation. The main advantage of this technique is that the severity of the brain damage can be easily controlled by manipulating the biochemical parameters such as velocity and the death of the impact on the CCI device. The CCI-induced focal cortical shatter injury is highly reproducible.

Proper stereotactic technique and the craniotomy procedure are major determinants in producing stable and reproducible CCI-induced brain injury. In general, investigator new to the procedure will struggle with stabilize the mouse onto the steriotexic frame and make the appropriate craniotomy. Demonstrating the procedure will be Ms.Jhih Shuan Lin, a lab technician from my lab.

Begin by confirming a lack of toe pinch reflex in the animal to ensure that it is properly anesthetized. Shave the mouse's head with electrical clippers in a caudal to rostral direction. But do not trim the whiskers.

Place the mouse onto the stereotaxic frame and carefully insert the ear bars into the ear canals, making sure that the mouse head is stabilized by both ear bars equally. Maintain anesthesia at one to 2%isoflurane for the duration of the surgery. Apply petroleum jelly to both eyes to prevent drying out during the surgery.

Then use sterile cotton swabs to disinfect the shaved head with betadine followed by 70%ethanol. Administer 100 microliters of 0.25%Bupivacaine subcutaneously with a 31 gauge insulin needle, and gently massage the injection site for better absorption. Make a longitudinal incision along the midline on the scalp with a scalpel or scissors.

Then use a hemostat to pull the skin off to the right side. Allow the exposed skull to dry for one minute. Then clean away any residual blood and tissues on the skull with a sterile cotton swab.

Check that the animal's head is level in the horizontal plane and identify the anatomical landmarks, Bregma and Lambda, marking both locations with a pencil. Ensure that the head of the animal is level in the rostral-caudal direction by measuring the Z coordinates of both Bregma and Lambda. Use the same procedure to horizontally position the head.

Use a 31 gauge insulin needle to identify the craniectomy site. Set the XY origin to Bregma and laterally move the needle three millimeters to the right. Mark this position as the site of craniectomy and draw a circle four millimeters in diameter on the skull with a pencil.

Cut along the pencil outlined circle with a high-speed micro drill with the trephine to create a four millimeter diameter open hole. Remove the bone flap with a tweezer and store it in ice cold, normal saline. Gently rinse the hole with saline before applying pressure on the brain surface to stop the bleeding.

On the CCI device, set the 2.5 millimeter diameter rounded impactor tip to an angle of 22.5 degrees. Zero the impact tip to the dural surface, and set the impact parameters on the control box to a velocity of four meters per second and a deformation depth of two millimeters. Retract the metal tip.

Discharge the piston to generate impaction on the brain. Then place a sterile cotton swab onto the injured area to stop bleeding. Replace the bone flap and secure it with dental cement.

Close the scalp with tissue adhesive. Place the mouse under the heat lamp in a clean recovery cage with bedding until full recovery. Proper stereotactic technique and craniectomy procedure are major determinants in producing stable and reproducible CCI-induced brain injury.

An ideal craniectomy procedure would cause minimal histological injury in the sham operated brain. Changes in brain damage and corpus callosum loss were observed at days 1, 3, 7, and 28 after CCI. In addition, unilateral brain atrophy on the contusion side was seen on day 28 post-CCI.

Neuroinflammation was revealed by Iba1-positive activated microglia and macrophage accumulation around the border of the contusion area. The intraparenchymal hemorrhage was also detectable by the Ly76-positive staining from days one to seven post-CCI. A mixture of red blood cells with ruptured and intact cell morphology was observed at day seven post-CCI, suggesting the RBCs were lysed at this stage.

This phenomenon was consistent with the observation that ferric iron deposition was detectable in the contusion region from days seven to 28 post-CCI. Activated microglia and macrophages were observed in the striatum far away from the contusion site from days three to 28 post-CCI, indicating corpus callosum and striatal injuries. This technique paves the ways for investigator to further explore brain hemorrhage-induced neuroinflammation interaction through understanding the response of the immune cells, such as microglia, after hemorrhage contusion.