A Structured Approach to Extubation in Mechanically Ventilated Rats

Summary

This study proposes a structured extubation approach tailored to rat models of mechanical ventilation (MV). The approach facilitates a smooth transition from MV to spontaneous breathing while minimizing extubation-related complications. Standardization of weaning, airway management, and post-extubation care enhances the reliability and reproducibility of experimental studies.

Abstract

Mechanical ventilation (MV) is widely used in critical care but is associated with various complications that affect both short- and long-term outcomes, including ventilator-induced lung injury, ventilator-induced diaphragm dysfunction, and ventilator-associated brain injury. These are key components of post-intensive care unit syndrome (PICS). Rodent models are vital for investigating the prolonged impacts and underlying mechanisms of MV, yet standardized post-extubation models remain limited. Extubation is a critical transition step that is often inconsistently performed in existing rat models, leading to failures and complications. This study presents a four-step structured extubation approach for Sprague-Dawley rat models of MV, designed to optimize the transition from MV to spontaneous breathing while minimizing extubation-related complications. The approach involved: (1) initiating weaning by reducing propofol sedation to 50% and assessing readiness via respiratory rate, chest displacement, arterial blood gases, and reflexes; (2) preparing for extubation through airway clearance and maintenance of oxygenation; (3) executing extubation with maintained sedation and controlled tube removal; and (4) providing post-extubation care involving continuous monitoring and oxygen support. Validated through 20 successful extubations following initial failures, this model offers a valuable platform for studying the long-term effects of MV, particularly in the context of PICS. This approach is optimized for 6 h of MV and is assumed to be extendable to other strains and longer ventilation durations, although such adaptations require careful validation and monitoring to ensure reliability and animal welfare.

Introduction

Mechanical ventilation (MV) remains a cornerstone of critical care, providing essential respiratory support for acute respiratory failure and serving as a vital adjunct during anesthesia for surgical procedures¹. However, MV is associated with a range of complications, such as ventilator-induced lung injury², ventilation-induced diaphragm and expiratory muscles dysfunction^3,4,5,6, and ventilator-associated brain injury^7,8. These complications contribute to extubation failure, prolonged stay in intensive care units (ICU) and hospitals, and increased mortality⁹. As ICU survival rates improve, attention has increasingly shifted toward the long-term consequences of MV, with post-ICU syndrome (PICS) emerging as a critical concern¹⁰. The pathophysiological mechanisms underlying PICS remain unclear, partly due to the lack of reliable animal models to capture the nuances of post-extubation recovery and long-term sequelae of MV¹¹.

Rat models have been pivotal in advancing the understanding of MV-induced injuries, offering scalability, reproducibility, and mechanistic insights. However, most existing models focus on the acute effects of MV, with tracheostomy being the predominant method for MV in experimental settings^12,13,14. While tracheostomy facilitates MV and weaning after it, it bypasses the upper airway, a critical anatomical and functional barrier. This increases the susceptibility to pulmonary complications such as ventilator-associated pneumonia^15,16. Additionally, the absence of nasal airflow in tracheostomy models disrupts brain network synchronization. This complicates investigations into cognitive and neurological outcomes^17,18. In contrast, endotracheal intubation /extubation models retain the integrity of the upper airway. They avoid the inherent disadvantages of tracheostomy and more closely resemble the clinical trajectory of weaning from MV.

However, existing extubation models often lack standardization, leading to considerable variability in the transition from MV to spontaneous breathing^19,20. This lack of structure introduces risks of extubation-related complications, including airway obstruction, respiratory distress, and aspiration, which may result in animal mortality. Such failures not only compromise the ability to investigate long-term outcomes but also render the initial MV model ineffective, wasting valuable time, resources, and financial investment. These issues underscore the critical need for a structured extubation approach to ensure successful post-extubation survival and robust long-term studies.

To address these challenges, a structured extubation approach was developed for Sprague-Dawley (SD) rat models that closely aligns with clinical practices for weaning from MV. This approach encompasses four key stages: approaching weaning, preparing for extubation, executing extubation, and post-extubation care. Key procedural recommendations include maintaining light-to-moderate sedation during weaning, specifically, reducing propofol infusion to 50% of the dose used during full MV -- to allow spontaneous breathing while preventing agitation. Extubation readiness should be assessed based on a stable respiratory rate of 60-70 breaths per minute and adequate spontaneous tidal volumes, which can be approximated using a simple, calibrated homemade scale. Full recovery of consciousness is not required prior to extubation. Propofol infusion should be discontinued at the time of extubation to further reduce the risk of intubation-induced agitation. By standardizing the extubation process, this approach minimized variability, avoided extubation-related complications, and enhanced survival rates. This model has been optimized for MV durations of up to 6 h; extending MV beyond this period may necessitate modified sedation protocols, incremental weaning strategies, and careful physiological monitoring to ensure survival and reliable outcomes. Although validated in SD rats, the protocol can be adapted for other strains by adjusting ventilation settings to account for strain-specific respiratory mechanics and varying susceptibilities to lung injury. With appropriate modifications, the structured approach may also support longer-term ventilation studies, enhancing both reproducibility and translational relevance across experimental models.

Protocol

The study focused on post-extubation behavioral testing and was approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (No. AEEI-2024-391). All rats aged 8 weeks (SD, male, 200-250 g) were acclimatized in the animal facility at Capital Medical University for 3 days prior to the experiment. The rat was housed under a controlled environment, including a 12-h light/dark cycle beginning at 8:00 AM, a temperature of 25 °C ± 1 °C, and humidity maintained at 50% ± 10 %. Food and water were provided ad libitum. All procedures involving anesthesia, suctioning, and blood handling were performed with appropriate safety measures, including the use of personal protective equipment, sterile techniques, and adherence to institutional biosafety protocols. All procedures were performed in compliance with the ARRIVE guidelines. All equipment used in the current study is presented in Figure 1. The list of reagents and the equipment used are provided in the Table of Materials.

1. Video laryngoscopy-assisted oral intubation

1. Prepare the intubation device.
   1. Use a custom-modified intubation device, adapted from an earwax removal device (scoop diameter 5.5 mm, curved into a tongue depressor). This device is equipped with a light source and camera, Bluetooth-linked to a smartphone for real-time visualization (Figure 2A).
2. Select an endotracheal tube.
   1. Choose a 16-G endotracheal tube based on the rat's weight.
3. Induce anesthesia (following institutionally approved protocols).
   1. Inject propofol (2 mg/100 g) via the tail vein. Confirm anesthesia by verifying the absence of visual and toe pinch responses. Monitor the distal skin color as an indicator of oxygenation status.
4. Position the rat.
   1. Secure the anesthetized rat on a small animal table. Fix limbs and incisors, and maintain body temperature at 37-38 °C using a small animal heating blanket.
5. Perform intubation.
   1. Gently pull the tongue to the right oral corner. Insert the intubation device at the base of the tongue and advance it toward the epiglottis.
   2. Lift the device tip to expose the glottis. Insert the endotracheal tube during inhalation (Figure 2-C).Once intubated, the rat will be connected to the ventilator.

2. Mechanical ventilation

1. Connect the oxygen concentrator.
   1. Adjust the appropriate oxygen concentration and connect with the ventilator (usually 80%).
2. Set ventilator parameters.
   1. Ventilate the rat for 6 h using a small animal ventilator with lung-protective settings: tidal volume: 4-6 mL/kg, PEEP: 0 cmH₂O.
3. Maintain anesthesia: Infuse propofol (4 mg/kg/h) via a syringe pump.
4. Monitor blood gases.
   1. Test arterial blood gases every 3 h using a blood gas analyzer. Continuously monitor the distal skin color between blood gas analyses as an indicator of oxygenation status.

3. Airway pressure endpoint control (APEC) extubation procedure

NOTE: After the planned duration of mechanical ventilation is completed, proceed with the four-step extubation protocol: (1) Approaching weaning, (2) Preparing extubation, (3) Executing extubation, and (3) Caring post-extubation (Figure 3). This systematic approach ensures a smooth transition to spontaneous breathing while minimizing extubation-related complications and preserving the integrity of the experimental model.

1. Approaching weaning
   1. Reduce propofol infusion: Decrease the propofol infusion rate by 50% to allow diaphragm reactivation while minimizing limb and head movements.
   2. Assess readiness for weaning: Check the following parameters before disconnecting the ventilator - Respiratory rate: 60-70 breaths/min; Chest displacement: ≥1 mm (measure using a steel needle placed on the xiphoid process); PaCO₂: 35-45 mmHg; pH: 7.35-7.45; Presence of whisker and tongue reflexes (optional).
      NOTE: The whisker and tongue reflexes were defined as binary indicators (absent or present). The whisker reflex refers to the involuntary movement or twitching of the vibrissae in response to light tactile stimulation. The tongue reflex was characterized by tongue withdrawal or movement upon gentle stimulation.
   3. Disconnect ventilator: Disconnect the ventilator once all criteria are met, and proceed to prepare the rat for extubation.
2. Preparing for extubation
   1. Ensure oxygenation: Use a self-made face mask to maintain adequate oxygenation (usually 80%), and use skin color as an indicator of oxygenation status.
   2. Clear airway secretions: Use a self-made suction device (shortened epidural catheter [~5.5 cm] connected to a 10 mL syringe). Control suction speed at ~2 mL/s for 3-4 s per session.
   3. Following adequate preparation and airway clearance, the procedure proceeds to the execution of extubation.
      NOTE: Typical secretion volume is <0.5 mL per suction. Suction is performed every 30 min during MV to minimize accumulation. At the time of extubation, the suction frequency was commonly 5-8 times, with an estimated amount of 2-4 mL in total.
3. Executing extubation
   1. Maintain sedation: Keep propofol at half-dose to prevent agitation during extubation.
   2. Remove endotracheal tube: Use A two-handed technique and stabilize the rat's head with the left index finger and thumb. Gently remove the tube with the right index finger and thumb.
   3. Discontinue propofol: Stop propofol infusion immediately after extubation.
      NOTE: Successful extubation should not result in hypoxemia or hypercapnia. And after successful extubation, diligent post-extubation care is essential.
4. Post-extubation care
   1. Monitor vital signs: Monitor respiratory rate, inspiratory amplitude, and distal skin color.
   2. Provide oxygenation: Use a self-made face mask to ensure a sufficient oxygen supply (usually ~80%).
   3. Prepare for re-intubation: Keep all intubation devices (Figure 1) on hand and ready for immediate use if needed.
      NOTE: Average observation time until righting reflex restoration is 48.5 min ± 10.4 min. Arterial blood gas analysis was performed at a median of 20 min, with PaO₂ of 73.2 mmHg  ± 4.6 mmHg and PaCO₂ of 48.3  mmHg ± 3.6 mmHg.

Results

This structured extubation approach was developed on four failed extubations and 20 subsequent successful extubations. In the first four attempts, extubation was performed based on the experience or individual judgment. Reasons for the four rats' failed extubation included agitation during extubation(n = 2), post-extubation respiratory insufficiency (n = 1), and airway obstruction by secretions (n = 1). These failures highlighted that optimal sedation, a clear airway, and sufficient oxyge...

Discussion

In this study, a structured APEC extubation approach was established for a rat model of MV, inspired by the first four failed extubations and the underlying causes. All 20 rats extubated using this approach succeeded and completed subsequent behavioral testing within the following three days. The approach, consisting of four key steps -- Approaching weaning, Preparing extubation, Executing extubation, and Post-extubation care -- was designed to ensure a smooth transition from mechanical to spontaneous breathing while min...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16G Endotracheal Tube	NJKEWBIO	NA	Endotracheal tube
Blood Gas Analyzer	Epocal Inc	EPOC reader	Monitor blood gases
Intravenous Catheter	Introcan Safety	4254074B	Indwelled in the arteries and veins
Micro-syringe Pump	SLGO Medical Technology	CP-1000	Infusion of drugs
Pet Oxygen Concentrator	Beijing zhongshidichuang Science and technology development Co.,Ltd	ZS-MV	Supply of oxygen
Small Animal Heating Blanket	Beijing zhongshidichuang Science and technology development Co.,Ltd	ZS-T	Maintain temperature
Small Animal Ventilator	NJKEWBIO	KW-100	Provide breathing