Name: anesthesia and intubation of preadolescent mouse pups for cardiothoracic surgery
Uploaded: 2022-06-02T14:30:00
Description: Watch this Scientific Journal Video about Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery at JoVE.com

This work was supported by NHMRC Program Grant [ID 1074386], a Leducq Transatlantic Network of Excellence in Cardiovascular Research grant [RMG], and a grant from the RT Hall Trust [RMG &#38; SEI].

All animal experiments described were approved by the Garvan/St Vincent&#39;s Hospital Animal Ethics Committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the ARRIVE guidelines, and all experiments were performed by an experienced small animal surgeon (JW) with guidance from a pediatric anesthetist (JJS).
1. Preparation of instruments
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	<li>On the day of surgery, set up specialized equipment for intubatio...

<ol>
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Currently, there are no well-documented methods for anesthesia and intubation of 10-day-old mice for cardiothoracic surgery. To this end, we have titrated ketamine/xylazine/atropine dosage regimens to body weight, whereby doses of 20/4/0.12 mg/kg, 30/4/0.12 mg/kg, and 50/6/0.18 mg/kg facilitated intubation of pups with low (3.15-4.49 g), mid (4.50-5.49 g), and high (5.50-8.10 g) body weight, respectively. Survival post-intubation correlated with body weight (59%, 70%, and 80% for low-, mid-, and high-weight groups, respe...

Murine models are invaluable tools in preclinical cardiothoracic research, in particular because of the ease with which genetically-engineered mouse lines can be generated, and also the ease with which the mice can be surgically manipulated to provide pathological disease models to allow, for example, the study of myocardial regeneration after cardiac injury1. In this regard, it is of interest that, unlike adult mice in which cardiomyocytes have withdrawn from the cell cycle, 0-2-day-old neonatal mouse hearts repair with minimal scarring after apical resection or induction of myocardial infarction2,3,4.&#160;In contrast, 7-day-old neonatal hearts regenerate incompletely with a higher incidence of scarring2,3. Since cardiomyocytes in the apex of the left ventricle retain proliferative capacity for up to 2 weeks after birth, mechanistic studies of regeneration after cardiac injury in 0-14-day-old mice may be informative for identifying therapeutic targets for regeneration of the injured adult heart5.
The development of mouse models of cardiac injury involves surgical manipulation under anesthesia. This requires that the thorax be opened to access the heart, which generally mandates intubation and mechanical ventilation. Mouse strain, body weight, and age influence sensitivity to anesthetics6. Adult mice can be anesthetized with a wide range of agents, a common regimen for intubation being ketamine/xylazine/atropine at 100/13/0.5 mg/kg6,7. Neonatal mice (0-7 days old) lack a centralized pain reflex, and can be effectively immobilized on ice and subjected to surgery without intubation6,8,9. Preadolescent (8-14-day-old) mouse pups cannot be anesthetized with hypothermia9,10; they require intubation for cardiothoracic surgery. There are no previous studies on cardiothoracic surgery in preadolescent mice less than 14 days old. In our experience, intubation of isoflurane-anesthetized preadolescent mice under 14 days of age is difficult. The recommended injectable anesthetic regimen reported for mice older than 7 days is 50-150 mg/kg ketamine and 5-10 mg/kg xylazine10. Preadolescent mice are still developing neurologically and their responses to drugs and drug metabolism are very different from adult animals6. This poses increased risk of fluid, electrolyte, and acid-base imbalance, as well as hypoglycemia and hypothermia due to not only their high metabolic rate, which rapidly depletes their limited energy stores, but also due to their thermoregulatory immaturity6,11,12. Thus, there is little information on anesthetic regimens that both facilitate intubation and maximize survival of preadolescent mice.
Here we empirically titrated dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups ranging in weight from 3-8 g to achieve a plane of anesthesia sufficient to allow endotracheal intubation for subsequent cardiothoracic surgery, while minimizing animal mortality. We also refined animal handling practices to reduce mortality from intubation, surgery, and post-surgical maternal cannibalism.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atipamezole (Antisedan)</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>ATIP I</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 600 mcg/mL</td>
			<td>Clifford Hallam Healthcare Pty Ptd</td>
			<td>1957699 PFIZER-0143386</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Livingstone International</td>
			<td>BU0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>TEMG I</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-optic light</td>
			<td>Leica&#160;</td>
			<td>3011350</td>
			<td>CLS 150X</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, LLC</td>
			<td>Version 9.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Intubation platform&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Any sturdy box (e.g. plastic tip box) with approximate dimensions 12 (L) x 8.5 x (W) x 7.5 cm (H)</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>ISOF 07</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>KETAI1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic intravenous cannula 24-gauge Polywin Safety&#160;</td>
			<td>BD Insyte&#160;</td>
			<td>CE0086</td>
			<td>19 mm length of plastic tubing (0.7 mm outer diameter) attached to a 21mm plastic female luer lock adaptor; total volume of annula 130 &#956;L</td>
		</tr>
		<tr>
			<td>Single lumen polyethylene tube</td>
			<td>Critchley Electrical Products Pty Ltd Auburn NSW</td>
			<td></td>
			<td>Outer diameter 0.61 mm, inner diameter 0.28 mm</td>
		</tr>
		<tr>
			<td>Small forceps</td>
			<td>F.S.T.</td>
			<td>NO 11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope (camera optional)</td>
			<td>Leica&#160;</td>
			<td>M651 (Leica IC80 HD camera)</td>
			<td>10x and 16x objective</td>
		</tr>
		<tr>
			<td>Suture 7-0 prolene</td>
			<td>Ethicon</td>
			<td>8708H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 9-0 polypropylene monofilament</td>
			<td>Ethicon</td>
			<td>2813</td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 Tabletop with Active Scavenging isoflurane anesthesia systm</td>
			<td>VetEquip</td>
			<td>901820</td>
			<td></td>
		</tr>
		<tr>
			<td>Vented 2-Liter plexiglass induction chamber</td>
			<td>VetQuip Pty Ltd</td>
			<td>942102</td>
			<td>25 cm (L) x 13 cm (W) x 11 cm (H)</td>
		</tr>
		<tr>
			<td>Warming lamp</td>
			<td>Brilant Lighting</td>
			<td>99223</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>XYLA Z 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

anesthesia and intubation of preadolescent mouse pups for cardiothoracic surgery

Murine surgical models play an important role in preclinical research. Mechanistic insights into myocardial regeneration after cardiac injury may be gained from cardiothoracic surgery models in 0-14-day-old mice, the cardiomyocytes of which, unlike those of adults, retain proliferative capacity. Mouse pups up to 7 days old are effectively immobilized by hypothermia and do not require intubation for cardiothoracic surgery. Preadolescent (8-14-day-old) mouse pups, however, do require intubation, but this is challenging and there is little information regarding anesthesia to facilitate intubation.&#160;Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality. Empirical titration of ketamine/xylazine/atropine dosage regimens to body weight indicated that the response to anesthesia of mouse pups of different weights was non-linear, whereby doses of 20/4/0.12 mg/kg, 30/4/0.12 mg/kg, and 50/6/0.18 mg/kg facilitated intubation of pups weighing between 3.15-4.49 g (n = 22), 4.50-5.49 g (n = 20), and 5.50-8.10 g (n = 20), respectively. Lower-body-weight pups required more intubation attempts than heavier pups (p &#60; 0.001). Survival post-intubation correlated with body weight (59%, 70%, and 80% for low-, mid-, and high-weight groups, respectively, R2 = 0.995). For myocardial infarction surgery after intubation, a surgical plane of anesthesia was induced with 4.5% isoflurane in 100% oxygen and maintained with 2% isoflurane in 100% oxygen. Survival post-surgery was similar for the three weight groups at 92%, 86%, and 88% (p = 0.91). Together with refinements in animal handling practices for intubation and surgery, and minimizing cannibalization by the dam post-surgery, overall survival for the entire procedure (intubation plus surgery) correlated with body weight (55%, 60%, and 70% for low-, mid-, and high-weight groups, respectively, R2 = 0.978). Given the difficulty encountered with intubation of 10-day old pups and the associated high mortality, we recommend cardiothoracic surgery in 10-day-old pups be restricted to pups weighing at least 5.5 g.

Anesthesia of 10-day-old mice. The10-day-old pups can be anesthetized with 4.5% isoflurane in 4-5 min; however, they recover from anesthesia in the process of preparation for intubation. Due to their small size, intubation under isoflurane anesthesia delivered by a standard nose cone is not feasible. We have previously used a ketamine/xylazine/atropine anesthetic regimen of 100/13/0.5 mg/kg, respectively, for cardiothoracic surgery in 15- and 21-day-old pups and adults4,</sup...

Watch this Scientific Journal Video about Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery at JoVE.com

Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

Cardiothoracic surgical models in mice &gt;7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality.

Murine surgical models play an important role in preclinical research. Mechanistic insights into myocardial regeneration after cardiac injury may be ...

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.

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