Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

Summary

Cardiothoracic surgical models in mice >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.

Abstract

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. 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 < 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.

Introduction

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 infarction^2,

Protocol

All animal experiments described were approved by the Garvan/St Vincent'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

1. On the day of surgery, set up specialized equipment for intubation of 10-day-old pups (Figure 1A,B). This includes a warming lamp, intubation platform, fiber-optic ligh....

Results

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 adults^4,

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Atipamezole (Antisedan)	Provet (NSW) Pty Ltd	ATIP I
Atropine 600 mcg/mL	Clifford Hallam Healthcare Pty Ptd	1957699 PFIZER-0143386
Betadine	Livingstone International	BU0520
Buprenorphine (Temgesic)	Provet (NSW) Pty Ltd	TEMG I
Fiber-optic light	Leica	3011350	CLS 150X
GraphPad Prism	GraphPad Software, LLC	Version 9.1.2
Intubation platform	-	-	Any sturdy box (e.g. plastic tip box) with approximate dimensions 12 (L) x 8.5 x (W) x 7.5 cm (H)
Isoflurane	Provet (NSW) Pty Ltd	ISOF 07
Ketamine 100 mg/mL	Provet (NSW) Pty Ltd	KETAI1
Plastic intravenous cannula 24-gauge Polywin Safety	BD Insyte	CE0086	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 μL
Single lumen polyethylene tube	Critchley Electrical Products Pty Ltd Auburn NSW		Outer diameter 0.61 mm, inner diameter 0.28 mm
Small forceps	F.S.T.	NO 11051-10
Surgical microscope (camera optional)	Leica	M651 (Leica IC80 HD camera)	10x and 16x objective
Suture 7-0 prolene	Ethicon	8708H
Suture 9-0 polypropylene monofilament	Ethicon	2813
V-1 Tabletop with Active Scavenging isoflurane anesthesia systm	VetEquip	901820
Vented 2-Liter plexiglass induction chamber	VetQuip Pty Ltd	942102	25 cm (L) x 13 cm (W) x 11 cm (H)
Warming lamp	Brilant Lighting	99223
Xylazine	Provet (NSW) Pty Ltd	XYLA Z 2