To begin, lay the eight to 10-week-old, anesthetized C57 black 6 mouse on the surgical bench. Using blunt forceps, pull out the tongue and hold it. Insert a laryngoscope into the mouth and visualize the vocal cord.
Insert a guide wire into a 20 gauge intravenous catheter. Then gently push the catheter into the trachea until its wing part is even with the nose tip. Connect the mouse to the ventilator, and place it in a supine position under a heat lamp.
Maintain the rectal temperature at 37 degrees Celsius. After shaving and disinfecting the surgical area, make a one centimeter skin incision on the left and right inguinal areas to access the femoral arteries. Then disinfect and ligate the distal femoral artery, and apply one drop of lidocaine.
Place a clip on the proximal femoral artery, and make a small cut on the artery distal to the clip. Insert a polyethylene 10 catheter into both the left and right femoral arteries. Inject 50 microliters of one to 10 heparinized saline into each arterial line.
To prevent clotting in the line, turn the mouse to the prone position, and mount it on a stereotaxic head frame. Connect three needle electrodes to the left arm, left leg, and right arm for electrocardiogram monitoring. Then shave the top of the head and sterilize it with three alternating rounds of iodine and alcohol swabs.
Make a 2.5 centimeter midline skin incision, and use four small retractors to expose the entire skull surface for brain imaging. Then place a Laser Speckle Contrast Imager above the head. Adjust the Laser Speckle Imager to see the baseline of cerebral blood vessels before cardiac arrest.
Before inducing cardiac arrest, fill a one milliliter plastic syringe with 26 microliters of the resuscitation cocktail stock solution. Once the body temperature reaches 37 degrees Celsius, adjust the oxygen meter to 100%to oxygenate the blood for two minutes. Then draw up to 200 microliters of blood through the right femoral artery into the plastic syringe containing the resuscitation cocktail.
To induce cardiac arrest, switch off the oxygen and increase the nitrogen to 100%to induce anoxia. Turn off the ventilator, isoflurane vaporizer, temperature controller, and nitrogen flow meter. Confirm the absence of brain blood flow during cardiac arrest.
By observing the laser speckle image, adjust the oxygen to 100%and turn on the ventilator eight minutes after cardiac arrest onset. Immediately infuse the withdrawn oxygenated blood mixed with the resuscitation cocktail into blood circulation through the right femoral artery in one minute to restore vitals such as heart rate and blood pressure. See the Laser Speckle Image to confirm successful reperfusion of the brain.
For postoperative recovery, remove the retractors from the head, apply 0.25%bupivacaine to the skin incision, and close the incision using a 6-0 nylon suture thread. Then apply antibiotic ointment on the closed skin incision surface. Induction of cardiac arrest in the mouse led to severe bradycardia within 45 seconds.
Following two minutes of an anoxia, the heart rate significantly reduced. The blood pressure decreased below 20 millimeters of mercury and the cerebral blood flow ceased. Also, the body temperature slowly dropped to about 32 degrees Celsius at the end of the cardiac arrest.
After injecting the blood resuscitation mixture, all vital functions started to recover. Laser Speckle Contrast Imaging confirmed the complete absence of blood flow in the brain during cardiac arrest, the photoacoustic images enabled the identification of changes in blood flow, structure, and oxygenation during the cardiac arrest procedure.
