Anesthesia and Intubation of Preadolescent Mouse Pups for Cardiothoracic Surgery
June 2nd, 2022
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.
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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Overview
This study addresses the challenges of intubating pre-adolescent mouse pups (10-day-old C57BL/6J) for cardiothoracic surgery. It presents a new protocol for anesthetic regimens that minimizes mortality during the intubation process.
Key Study Components
Research Area
- Cardiothoracic surgical models
- Anesthesia protocols in small mammals
- Mouse physiology in surgical settings
Background
- Existing limitations in intubation techniques for young mice
- Need for increased success rates in cardiothoracic surgery
- Importance of maintaining animal welfare during procedures
Methods Used
- Development of anesthetic regimens (ketamine/xylazine/atropine)
- Experimentation with C57BL/6J mouse pups
- Endotracheal intubation procedure and monitoring
Main Results
- Successful intubation of 10-day-old pups using specific dosages
- Lower mortality rates with optimized anesthetic regimens
- Demonstration of procedure efficacy by a skilled operator
Conclusions
- This study establishes a reliable method for anesthetic management in young mouse pups undergoing surgery.
- The findings enhance surgical methodologies and animal welfare in research settings.
Frequently Asked Questions
Executive Industry Relevance
Reliable anesthesia and intubation protocols for preadolescent mouse pups are critical for generating robust cardiothoracic surgical models in preclinical research. This method enables mechanistic studies of myocardial regeneration in a developmentally relevant system, supporting predictive confidence in early-stage target validation. Standardized intubation and anesthesia regimens reduce procedural variability and mortality, enhancing the translational value of murine cardiac injury models for biopharma R&D portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables mechanistic interrogation of myocardial regeneration in juvenile murine models.
- Supports functional target validation by providing access to a proliferative cardiomyocyte system.
- Facilitates biological de-risking by minimizing anesthesia-related confounders in surgical studies.
- Improves predictive confidence for early-stage cardiac therapeutic hypotheses.
Screening & Assay Development
- Standardizes animal handling and anesthesia for reproducible cardiothoracic surgical workflows.
- Provides validated protocols for consistent intubation and surgical preparation in young mice.
- Enables quantitative assessment of survival and procedural success across weight groups.
- Prepares robust preclinical models for downstream compound evaluation and screening.
Translational & Preclinical Research
- Aligns preclinical models with disease-relevant regenerative biology in early postnatal myocardium.
- Ensures continuity from discovery through preclinical validation by reducing procedural mortality.
- Supports risk-adjusted advancement decisions based on standardized survival metrics.
- Enhances translational biomarker studies by enabling consistent surgical injury models.
Pipeline & Workflow Integration
This protocol integrates into the early discovery-to-preclinical continuum by enabling reliable cardiothoracic surgery in preadolescent mice, a key inflection point for regenerative cardiac research.
- Discovery Biology: Facilitates hypothesis testing on myocardial regeneration and injury response in a developmentally relevant context.
- Screening: Provides reproducible, quantitative outputs for survival and procedural success, supporting assay readiness.
- Analytics: Enables statistical comparison of survival and intubation success across defined weight cohorts.
- Translational Research: Bridges early mechanistic studies with preclinical validation in regenerative cardiac models.
- Enterprise Reuse: Establishes a reusable protocol for diverse cardiothoracic and regenerative studies in juvenile mice.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cardiac regeneration studies.
- Operational Value: Delivers standardized, scalable, and reproducible anesthesia and intubation workflows.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by reducing procedural attrition.
- Portfolio Impact: Enables risk-adjusted prioritization of regenerative cardiac programs based on robust preclinical data.
Implementation Considerations
- Requires expertise in murine anesthesia and intubation, with proficiency gained through practice on multiple litters.
- Needs specialized equipment, including custom cannulas and ventilators adapted for small animal models.
- Demands rigorous cross-team standardization of anesthesia dosing and animal handling protocols.
- Must adapt dosage regimens to body weight to minimize mortality and procedural variability.
- Limited to pups above 5.5 g for optimal survival, restricting applicability to specific developmental windows.
Biopharma FAQs
Chapters in this video
0:05
Introduction
1:00
Preparation of Instruments and Intubation of 10-Day-Old Mice
3:09
Myocardial Infarction Surgery of 10-Day-Old Mice
5:13
Post-Surgery Assessment of Infarct Size
7:04
Results: Anesthesia Regimen, Number of Intubation Attempts, and Post-Procedure Survival of 10-Day-Old Mouse Pups
8:58
Conclusion
