Our protocol is the first documented method for anesthesia and endotracheal intubation of pre-adolescent mouse pups For cardiothoracic surgery. We have developed anesthetic regimens, specialized equipment, and specific handling practices that allow endotracheal intubation of 10=day old C57 Black Six J mouse pups for cardiothoracic surgery while minimizing animal mortality. We anticipate that an operator experienced in adult intubations can achieve proficiency in pre-adolescent intubation after practicing on 10 litters of seven to eight pups Demonstrating the procedure will be Dr.Jianxin Wu, a senior staff scientist from our laboratory.
On the day of the experiment, set up specialized equipment for intubation of 10-day old pups as described in the manuscript. For the surgery, use a cannula consisting of a 19 millimeter long plastic tubing attached to a 21 millimeter plastic female lure lock adapter. Stiffen the cannula's tubing by inserting a piece of copper wire via the lure lock adapter.
Secure the anesthetized pup supine to an intubation platform. Before intubation of the anesthetized pup, check the depth of anesthesia by the paw pinch reflex. The paw pinch reflex must be present but markedly reduced from that of a conscious animal.
Hold the pup's tongue with small forceps and use a laryngoscope fashioned out of a piece of copper wire and a flexible fiber optic light to expose the glottis and vocal cords. Tilt the stiffened cannula so that the lure lock end is slightly lower than the tip, and as soon as the vocal cords separate insert the cannula and advance it until the lure lock adapter is just outside the mouth. Then immediately remove the wire.
Assess the depth of anesthesia after the intubation by the ability of the animal to breathe spontaneously. Transfer the intubated pup to a warming pad set at 37 degrees Celsius. Confirm successful tracheal intubation of spontaneously breathing pup by briefly blocking the intubation catheter to check that this prevents chest movement.
Connect the endotracheal cannula to a ventilator, delivering 100%oxygen at a flow rate of one liter per minute. The procedure should take less than 15 seconds to minimize rebreathing. After inducing a surgical plane of anesthesia for the surgery, confirm tracheal intubation by checking that the frequency of chest wall movement equals that of the ventilator.
Place the pup under a surgical microscope with 10X and 16X objectives for the myocardial infarction surgery. After disinfecting the skin, make a horizontal skin incision between the third and fourth rib in the left lateral wall of the chest using a scalpel. With the help of fine forceps, open the thorax by blunt dissection of the in intercostal space and use a retractor to keep the space open.
Induce a myocardial infarction by ligating the left coronary artery distal to the left atrial appendage with a 9-0 polypropylene monofilament suture. After the 10 minutes of the infarction surgery, close the skin with a 7-0 suture. Clean the blood with 70%ethanol or saline and disinfect the incision with Betadine.
Allow the animal to recover and ensure spontaneous breathing resumes within a few minutes. Then return the pup to the warmed pre-oxygenated chamber and monitor continuously during recovery. As the righting reflex is regained, extubate the pup.
Gently rub the pup with home cage bedding. Keep the pup warm and check that breathing is regular and the pup is capable of spontaneous movement. Return the dam to the cage.
Then return all pups when they have fully recovered from anesthesia. House the dam and pups overnight in a cage placed half on a warming pad set at 37 degrees Celsius. On the third day post-surgery, secure the anesthetized pup in a supine position on a warming pad by taping the tail.
Place a thread over the incisors and tape the pup into a position and keep the head extended into a nose cone. Disinfect the skin with 70%ethanol. Using fine scissors, make a one centimeter incision in the skin over the right common carotid artery along the trachea and cannulate the exposed vessel using a single lumen polyethylene tube attached to a 26 gauge needle to administer 0.2 milliliters of heparinized saline for one minute after causing a cardiac arrest as described in the manuscript, dissect the right jugular vein via the previous incision and transect it.
Perfuse the heart with 0.2 milliliters of PBS and then with 0.1 milliliters of 0.2%Alcian Blue to stain the non-infarcted remote myocardium. Check successful perfusion, evidenced by the washing out of blood, PBS and then Alcian Blue via the jugular vein. When done, open the thorax and excise the heart by dissecting the surrounding connective tissue and vessels to release the heart.
Rinse the heart in PBS, then remove the atria and photograph the heart with a camera mounted on a surgical microscope using a 10X subjective. In the preliminary experiments, the anesthesia regimen was optimized for 10-day old pups. A dose of 50 milligrams per kg Ketamine, six milligrams per kg Xylazine, and 0.18 milligrams per kg of atropine resulted in a sufficient depth of anesthesia to allow endotracheal intubation of spontaneously breathing pups weighing 5.5 to 7.30 grams.
As the lighter pups could not tolerate the higher dose of anesthesia, decreasing the dosage to 30 milligrams per kg ketamine, four milligrams per kg xylazine, and 0.12 milligrams per kg atropine enabled the intubation of the pups weighing 4.50 to 5.49 grams. In comparison, further reduction of the ketamine dosage to 20 milligrams per kg enabled the intubation of pups weighing 3.15 to 4.49 grams. The table also shows the number and percentage of intubated pups that proceeded to surgery.
The pups with lower body weights were more difficult to intubate than heavier pups and required more attempts which resulted in a lesser survival percentage of the pups than the highest weight group. Moreover, a larger number of the intubated pups from the highest weight group proceeded to the myocardial infarction surgery than the low and mid-weight group. The survival two days after myocardial infarction surgery was consistent between the different weight groups at 86%to 92%Depth of anesthesia is critical for intubation and survival.
If anesthesia is too light, intubation is difficult. If too deep, pups will stop breathing spontaneously either during or after intubation. Our anesthesia intubation protocol will allow models requiring cardiac thoracic surgery to be established in pre-adolescent mice, allowing, for example, studies on the lungs, esophagus and heart during this period.
