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Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery

Published: June 2, 2022 doi: 10.3791/64004
Jianxin Wu*1, Amy M. Nicks1,2, Justin J. Skowno3,4, Michael P. Feneley1,2, Robert M. Graham1,2, Siiri E. Iismaa*1,2
* These authors contributed equally

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 infarction2,3,4. 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.

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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/g13,14.

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 with 70% ethanol. 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 3rd 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.

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Representative 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 adults4,7. In preliminary experiments, that included oxygen supplementation, it was found that the recommended injectable regimen of 50-150 mg/kg ketamine and 5-10 mg/kg xylazine10 resulted in an unacceptable mortality in 10-day-old pups. Given the inverse correlation between body weight and litter size of 10-day-old pups (R2 = 0.250, p < 0.0001; Figure 2), we titrated the anesthetic regimen according to body weight groupings. Reducing the ketamine/xylazine/atropine dosage to 50/6/0.18 mg/kg, respectively, resulted in a sufficient depth of anesthesia to allow endotracheal intubation of spontaneously breathing pups weighing 5.5-8.10 g (Table 1), but this dose was not tolerated by lighter pups. Reducing the ketamine/xylazine/atropine dosage to 30/4/0.12 mg/kg, respectively, enabled intubation of pups weighing 4.50-5.49 g, while further reduction of the ketamine dosage to 20 mg/kg enabled intubation of pups weighing 3.15-4.49 g (Table 1). Table 1 shows the number and percentage of intubated pups that proceeded to surgery; however, it is difficult to extract from this data anesthesia-related mortality from mortality associated with too many intubation attempts. In the interest of reducing animal wastage, we did not specifically quantitate anesthesia-related mortality.

Intubation of 10-day-old mice. Outcomes were best when intubation was achieved after only one or two attempts. Pups with a lower body weight were more difficult to intubate than heavier pups and required more attempts (p < 0.001; Table 1). Survival post-intubation correlated with body weight with 59%, 70%, and 80% survival for low-, mid-, and high-weight groups, respectively (R2 = 0.995, p = 0.04; Table 1).

Myocardial infarction surgery of 10-day-old mice. Pups were monitored for 2 days after surgery. There were no signs of pain post-surgery. Of the pups that did not survive to follow-up at 48 h (Table 1), one from the low-weight group died 6 h after surgery, one pup from each of the mid- and high-weight groups died before being placed back with the dam, and one pup from each of the mid- and high-weight groups were cannibalized by the dam within 16 h of surgery, with small body parts or nothing remaining the next morning. Survival 2 days after myocardial infarction surgery was consistent between the different weight groups at 86%-92% (p = 0.91; Table 1). Infarcted myocardium, as assessed 2 days post-surgery by Alcian-blue perfusion of the heart, was evident by clear demarcation of stained, non-infarcted (blue) from ischemic (unstained) tissue, distal to the ligation (Figure 1E).

Overall survival for the entire procedure (intubation plus surgery) correlated with pup body weight at 55%, 60%, and 70% for low-, mid-, and high-weight groups, respectively (R2 = 0.978, Table 1), although this correlation did not achieve statistical significance (p = 0.09).

Figure 1
Figure 1: Endotracheal intubation of a 10-day-old C57BL/6J mouse pup. (A) Intubation set-up showing large warming lamp (WL), intubation platform (IP), and flexible fiber-optic lighting (FL) used to aid visualization of the vocal cords at the time of intubation. (B) Forceps, laryngoscope, 24-gauge cannula that is used as an endotracheal tube, and a piece of copper wire that is inserted into the endotracheal tube via the luer lock adaptor to stiffen the cannula during intubation (scale bar = 1 cm). (C) The anesthetized pup is secured supine by taping the tail and front limbs onto the intubation platform (12 cm (L) x 8.5 cm (W) x 7.5 cm (H)). A thread placed over the incisors is used to extend the head and is taped in position. (D) The fiber-optic light is placed over the neck to trans-illuminate the trachea just below the vocal cords. The tongue is held with small forceps, and then movement of the vocal cords is visualized by exposing the glottis with the laryngoscope. The endotracheal tube is inserted into the trachea while the vocal cords are open. (E) Photograph of a representative mouse pup heart perfused with Alcian blue (frontal view with the base of the heart at the top and apex at the bottom, and atria removed) 48 h post-ligation (black suture, black arrow) of the left coronary artery taken under a surgical microscope (10x objective) mounted with a camera. Non-infarcted myocardium is stained blue, infarcted myocardium at the apex is unstained and pale; scale bar = 100 µm. This figure has been modified from17. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Body weight of 10-day-old C57BL/6 pups is inversely correlated with litter size. Pups were from litters ranging in size from 4-10. The average C57BL/6 litter size is seven18. Data were analyzed by simple linear regression, with p < 0.05 being considered significant. This figure has been modified from17. Please click here to view a larger version of this figure.

Anesthesia regimen: ketamine/ xylazine/ atropine (mg/kg); given in 10 mL/g body weight, ip Body weight, g Number of pups studied Intubation attempts (A, 1-2; B, 3-4 or C, >4) and number of pups intubated, n (%) Intubated pups proceeding to surgery, n (%) Survival two days post-surgery, n (%) Overall survival after intubation plus surgery, n (%)
A B C
20/4/0.12 3.15 - 4.49 22 8 (36) 9 (41) 5 (23) 13 (59) 12 (92) 12 (55)
30/4/0.12 4.50 - 5.49 20 13 (65) 5 (25) 2 (10) 14 (70) 12 (86) 12 (60)
50/6/0.18 5.50 - 7.30 20 13 (65) 3 (15) 4 (20) 16 (80) 14 (88) 14 (70)
p (Chi-square test) p<0.001 p=0.91
R2 (Correlation coefficient,  0.995, 0.978,
p value) p=0.04 p=0.09

Table 1: Anesthesia regimen, number of intubation attempts, and post-procedure survival of 10-day-old mouse pups. Data were analyzed by Chi-squared test, with p < 0.05 being considered significant.

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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, respectively. 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 animals weighing at least 5.50 g. A limitation of this intubation technique is that it is dependent on the skill and experience of the operator, and how quickly they can learn. However, it is anticipated that an operator experienced in adult intubations can achieve proficiency in preadolescent intubation after practicing on 10 litters of seven to eight pups. Another limitation is that the overall pup survival after intubation and myocardial infarction surgery ranged from 55% (lowest body weight group) to 70% (highest body weight group). Nevertheless, this is similar to the 60%-70% survival reported for 1-day-old pups, which require no intubation when subjected to myocardial infarction after immobilization on ice8.

We found 10-day-old mouse pups of different weights had a non-linear response to the ketamine/xylazine/atropine anesthetic regimen. This may reflect the developmental differences in a number of important areas. Basal metabolic rate scales allometrically to the three-quarter power of mass, from single cells to mammals19. This would influence drug disposition in the animals in the study, which varied in weight by two-and-a-half times. The maturity of drug metabolism or detoxification mechanisms is another factor that changes rapidly in the immediate postnatal period, as are mechanisms influencing free drug availability, such as protein binding20. Pharmacokinetic differences may not be the only explanation for non-linear drug-effect relationships, as differences in pharmacodynamic responses to sedative agents are also possible6. The use of oxygenation after intraperitoneal injection of anesthetics and prior to intubation likely improved the safety of the procedure, as has been noted recently for adults21. Further adjustments in dosage, particularly for the lowest body weight group, may improve survival.

The depth of anesthesia was critical for successful intubation. Intubation was difficult if the plane of anesthesia was too light, and if too deep, pups stopped breathing spontaneously, either during intubation or after intubation while being ventilated with oxygen. Handling of pups also sometimes caused breath-holding, especially during intubation. If breathing stopped during intubation, stimulation of the foot or tail, or returning pups to the warmed oxygen-filled chamber, was critical to restore regular breathing. Intubation was re-attempted when the pup resumed regular breathing. If breathing stopped after intubation, the animals were ventilated for up to 10 min with oxygen. If spontaneous breathing resumed during this time, the animals proceeded to surgery. However, we found that if spontaneous breathing was not restored within this time, pups did not recover from anesthesia or, if subjected to surgery, died during the recovery period.

Given the high metabolic rate of 10-day-old pups, it is best to limit depletion of energy stores by separating the dam from its pups for as short a time as possible and, thus, restricting the number of surgical operations to four or five pups per litter per day over a maximum period of 5-6 h. To reduce maternal cannibalism of pups that had undergone surgery, any littermates that did not undergo surgery were removed to foster mothers or culled before return of the dam to the cage. Our handling practices to reduce mortality from post-surgical maternal cannibalism were similar to those that have been reported for neonates9.

In conclusion, our feasibility study suggests that an injectable ketamine/xylazine/atropine anesthetic regimen considerably lower than that used for older mice is required to minimize mortality from intubation of 10-day-old mouse pups for subsequent cardiothoracic surgery, as are specific handling practices to reduce mortality from intubation, surgery, and post-surgical maternal cannibalism.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

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 & SEI].

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

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References

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Cite this Article

Wu, J., Nicks, A. M., Skowno, J. J., Feneley, M. P., Graham, R. M., Iismaa, S. E. Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery. J. Vis. Exp. (184), e64004, doi:10.3791/64004 (2022).More

Wu, J., Nicks, A. M., Skowno, J. J., Feneley, M. P., Graham, R. M., Iismaa, S. E. Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery. J. Vis. Exp. (184), e64004, doi:10.3791/64004 (2022).

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