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^2,3,4. In contrast, 7-day-old neonatal hearts regenerate incompletely with a higher incidence of scarring^2,3. 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/kg^6,7. Neonatal mice (0-7 days old) lack a centralized pain reflex, and can be effectively immobilized on ice and subjected to surgery without intubation^6,8,9. Preadolescent (8-14-day-old) mouse pups cannot be anesthetized with hypothermia^9,10; 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 immaturity^6,11,12. 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.

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 light, small forceps, a laryngoscope fashioned out of a piece of 0.02 mm diameter copper wire (60 mm in length with the end of the wire fashioned into a 3 mm diameter circle at an angle of 175°; Figure 1B), and a 24-gauge plastic intravenous (i.v.) cannula, which is used as an endotracheal tube.
2. Ensure that the cannula consists of a 19 mm long plastic tubing (0.7 mm OD) attached to a 21 mm plastic female luer lock adaptor (Figure 1B). Stiffen the tubing of the cannula by inserting a piece of copper wire via the luer lock adaptor. Use a cannula with a total volume of 130 μL for a mouse with tidal volume of ~8 μL/g^13,14.

2. Anesthesia of 10-day-old mice

1. On the day of surgery, remove the dam from a cage of 10-day-old C57BL/6J pups and place the cage on a warming pad (37 °C).
2. Anesthetize pups by 10 μL per g body weight intraperitoneal injection using a 0.5 mL insulin syringe and 29 G needle with ketamine/xylazine/atropine in the ratios detailed in Table 1 for different weight groupings.
3. Immediately after injection, place the pup into a warmed (37 °C) plexiglass chamber that has been pre-oxygenated with 100% oxygen.

3. Intubation of 10-day-old mice

1. After 3-4 min of oxygenation, transfer the pup to a platform for intubation essentially as for adult mice. Perform this with the pup in the supine position (Figure 1C) or suspended at a 45° angle¹⁵. Maintain temperature with a warming lamp.
   1. Before intubation, assess the depth of anesthesia by the paw pinch reflex. For optimal intubation, the paw pinch reflex must still be present but markedly reduced from that of a conscious animal.
2. After securing the anesthetized pup supine to an intubation platform (Figure 1C), hold the tongue with small forceps and use a laryngoscope fashioned out of a piece of copper wire (Figure 1B) to expose the glottis and vocal cords. Aid visualization of the vocal cords by trans-illumination with a flexible fiber-optic light (Figure 1D).
3. Using a stiffened cannula, tilt the cannula so that the luer lock end is slightly lower (~10°) than the tip, and as soon as the vocal cords separate, insert the cannula and advance it until the luer lock adaptor is just outside the mouth. Remove the wire immediately after intubation.
   NOTE: No resistance during intubation is expected in mice of this age unless the cannula is advanced too far, and resistance is felt from the carina.
   1. Assess the depth of anesthesia after intubation by the ability of the animal to breathe spontaneously. Confirm successful tracheal intubation of spontaneously breathing pups by briefly blocking the intubation catheter to check that this prevents chest movement.
4. Immediately transfer the intubated pup to a warming pad (37 °C) and connect the endotracheal cannula to a ventilator delivering 100% oxygen at a flow rate of 1 L/min with 30 μL/stroke, 40 μL/stroke, or 50 μL/stroke for 3.15-4.59 g, 4.50-5.49 g, or 5.50-8.10 g pups, respectively, and 150 strokes/min.
5. Perform these procedures rapidly, within <15 s to minimize re-breathing.

4. Myocardial infarction surgery of 10-day-old mice

1. To induce a surgical plane of anesthesia for surgery, switch the gas flowing into the ventilator from 100% oxygen to 4.5% isoflurane in oxygen (the isoflurane concentration being determined by a vaporizer) for 4-5min.
   1. After switching to isoflurane, confirm tracheal intubation again by checking that the frequency of chest wall movement equals that of the ventilator. Loss of spontaneous breathing followed by absence of a tail or paw pinch reflex indicates that a surgical plane of anesthesia has been reached (after 4-5 min).
2. Maintain anesthesia with 2% isoflurane in oxygen.
3. Perform myocardial infarction surgery under a surgical microscope (10x and 16x objective) as described in¹⁶.
   1. Disinfect the skin in a circular motion several times with alternating rounds of an iodine-based or chlorhexidine-based scrub and 70% alcohol. Using fine scissors, make a horizontal skin incision between the third and fourth rib (fourth intercostal space) in the left lateral wall of the chest. Using fine forceps, open the thorax by blunt dissection of the intercostal space and use a retractor to keep the space open.
   2. Induce a myocardial infarction by ligation of the left coronary artery just distal to the left atrial appendage with 9-0 polypropylene monofilament suture. After the ~10 min infarction surgery, close the skin with 7-0 prolene and disinfect the incision with betadine. Clean the pup of blood with 70% ethanol or saline.
      NOTE: Ligation of the left coronary artery in preadolescent pups is essentially bloodless, as it is with adult ligations.
   3. Administer one after the other with a 0.5 mL insulin syringe and 29 G needle: atipamezole (1-5 mg/kg, 10 μL, intraperitoneal) for rapid recovery from sedation, analgesia (buprenorphine, 0.075 mg/kg, 10 μL, subcutaneous), and saline (50 μL, intraperitoneal).
4. Allow animals to recover by discontinuing the isoflurane. Ensure spontaneous breathing resumes within a few minutes thereafter.
   1. Return the pup to the warmed pre-oxygenated chamber and monitor continuously during recovery until the righting reflex is regained, at which point extubate the pup.
   2. Gently rub the pup with home cage bedding, keep the pup warm, check that breathing is regular, and that the pup is capable of spontaneous movement. This will reduce post-surgery cannibalism by the dam.
5. Return the dam to the cage when all pups have fully recovered from anesthesia.
   NOTE: The overall time taken for preparation, anesthesia, intubation, surgery, and recovery of one pup can range from 40-60 min.
6. House dam and pups overnight in a cage placed half on/half off a 37 °C warming pad.

5. Post-surgery assessment of infarct size

1. On the 3^rd day post-surgery, anesthetize pups by placing them in a plexiglass chamber pre-equilibrated with 4.5% isoflurane in oxygen at 1 mL/min flow rate.
2. Once a surgical plane of anesthesia has been reached (after 4-5 min), assessed by the paw pinch reflex, remove the pup from the chamber and secure in supine position on a warming pad by taping the tail.
3. Place a thread over the incisors and tape into position to keep the head extended and place the head into a nose cone connected to a ventilator delivering 4.5% isoflurane in oxygen at 200 µL/stroke, 150 strokes/min. Maintain a surgical plane of anesthesia with 2% isoflurane in oxygen.
4. Disinfect the skin with 70% ethanol. Using fine scissors, make a 1 cm incision in the skin over the right common carotid artery along the trachea and cannulate the exposed vessel using a single lumen polyethylene tube (OD 0.61 mm, ID 0.28 mm) to administer 0.2 mL of heparinized saline (200 U) for 1 min to prevent blood clotting.
5. Increase isoflurane to 4.5% in oxygen for 1 min before rapidly administering 0.2 mL of 3.3 M KCl within 2 s to arrest the heart in diastole.
6. Dissect the right jugular vein via the same incision and transect it. Perfuse the heart with 0.2 mL of phosphate-buffered saline (PBS), and then perfuse with 0.1mL 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.
7. Open the thorax and excise the heart by dissecting the surrounding connective tissue and vessels to release the heart. Rinse the heart in PBS, remove the atria if desired, and photograph the heart with a camera mounted on a surgical microscope using a 10x objective.

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