Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

Although donation after donor brain death is the main source for solid-organ transplantation, the irreversible loss of brain function induces pathophysiological changes that lead to a systemic inflammatory response. This murine model of brain-death induction allows the use of a large variety of analytical tools and knockout models to study the pathways of brain-death regulation. For arterial catheterization after confirming a lack of response to pedal reflex and removing the upper abdominal hair, disinfect the exposed skin with 70%ethanol and use dissecting scissors to make a midline incision in the skin.

Using forceps, blunt dissect the submandibular glands and neck muscle tissue and separate the tissues to expose the common carotid artery. Place three 8-0 silk ligatures beneath the right common carotid artery and place a clamp on the proximal ligature. Bring tension in the artery so that the flow is suspended and close the most distal ligature.

Insert a 26-gauge arterial catheter through a small preformed skin hole on the cranial aspect of the incision. Squeeze the catheter with the forceps if the lumen is too large to reduce the backflow and secure the catheter with all three sutures, then use a single 5-0 monofilament nonabsorbable to fix the catheter to the skin near the preformed skin hole. To perform a tracheostomy, use forceps to blunt dissect the pretracheal musculature and place two 8-0 silk ligatures beneath the trachea.

Insert the ventilation tube between two tracheal cartilages to avoid unilateral ventilation and secure the tube with both prepared ligatures, then close the skin with a 6-0 monofilament nonabsorbable running suture and ventilate the mouse with a frequency of 150 per minute and a tidal volume of 200 microliters. To induce brain death in the experimental animal, arrange the mouse to the prone position and, holding the skin with forceps, use surgical scissors to remove the skin from the skull. Drill a one-millimeter caliper borehole perimedially above the left parietal cortex and use blunt forceps to penetrate the final tissue bridge of the skull.

After removing any sharp edges, insert a balloon catheter prefilled with saline with all of the air evacuated. When the catheter is entirely within the cranial cavity, use a syringe pump to begin inflating the catheter at approximately 0.1 millimeters per minute over a period of 10 to 15 minutes. When brain death occurs, stop the inflation of the balloon catheter and place a heating blanket over the mouse to avoid hypothermia.

After brain death has been confirmed, monitor and document the blood pressure regularly, infusing 100 microliters of saline every 30 minutes to stabilize the blood pressure of the animal. After four hours of brain death, exclude any animals without beating hearts and harvest the mouse organs and tissues of interest according to standard protocols. After brain-death induction, the blood pressure exhibits an initial hypertensive peak followed by a prolonged hypotensive phase.

Another well-established observation is that brain-death induction leads to activation of the immune system. Indeed, after four hours of brain death in this model, an organ-specific upregulation of immune markers is observed at the mRNA level. After a predefined period of brain death, the organs may be harvested for their direct analysis or for subsequent organ transplantation.

This model will enable in-depth studies into the influence of brain-death induced injury and has already revealed new insights into complement activation and leukocyte migration.