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
<ol>
	<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 In this regard, it is of interest that, unlike adult mice in which cardiomyocytes have withdrawn from the cell cycle, 0-2-day-old neonatal mouse hearts repair with minimal scarring after apical resection or induction of myocardial infarction2,3,4.&#160;In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 ...

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