Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Summary

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Abstract

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.

We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

Introduction

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients¹. It is characterized by both diffuse alveolar epithelial and endothelial injuries, leading to increased permeability and pulmonary edema, altered alveolar fluid clearance (AFC), and worsened respiratory distress². The resorption of alveolar edema and recovery from ARDS require epithelial fluid transport through the alveoli to remain intact, which suggests that a therapy improving AFC could be useful^3,4. Although lung-protective ventilation and a restrictive strategy for intravenous fluid therapy have proven beneficial in improving outcomes^2,5, they are still associated with high mortality and morbidity⁶. Therefore, there is an urgent need to develop effective therapies for the syndrome and to better understand the precise mechanisms through which such therapies might work.

Halogenated anesthetics, such as isoflurane or sevoflurane, have been widely used for general anesthesia in the operating room. Sevoflurane is associated with decreased inflammation in the lungs of patients undergoing thoracic surgery and with a decrease in postoperative pulmonary complications, such as ARDS⁷. Similar results have been found in a meta-analysis of patients after cardiac surgery⁸. Halogenated volatiles also have a bronchodilatory effect^9,10 and perhaps some properties that protect several organs, such as the heart^8,11 and the kidneys^12,13,14. Recently, there has been growing interest in the clinical use of inhaled anesthetics as sedatives in the intensive care unit (ICU). Both animal and human studies support the protective effects of pretreatment with halogenated agents before prolonged ischemia of the liver¹⁵, the brain¹⁶, or the heart¹¹. Halogenated agents also have potential pharmacokinetic and pharmacodynamic advantages over other intravenous agents for the sedation of critically ill patients, including a rapid onset of action and fast offset due to little accumulation in tissues. Inhaled halogenated agents decrease intubation times in comparison with intravenous sedation in patients undergoing cardiac surgery¹⁷. Several studies support the safety and efficacy of halogenated agents in the sedation of ICU patients^18,19,20. In experimental models of ARDS, inhaled sevoflurane improves gas exchange^21,22, reduces alveolar edema^21,22, and attenuates both pulmonary and systemic inflammation²³. Isoflurane also ameliorates lung repair after injury by maintaining the integrity of the alveolar-capillary barrier, possibly by modulating the expression of a key tight junction protein^24,25,26. In addition, mouse macrophages that were cultured and treated with isoflurane had better phagocytic effects on neutrophils than macrophages that were not treated with isoflurane²⁷.

However, the precise biological pathways and mechanisms accounting for the lung-protective properties of volatile anesthetics remain largely unknown to date, requiring further investigation¹⁸. Additional studies are also warranted to investigate the precise effects of sevoflurane on lung injury and to verify whether experimental evidence can be translated to patients. The first randomized control trial from our team found that the administration of inhaled sevoflurane in patients with ARDS was associated with oxygenation improvement and decreased levels of both pro-inflammatory cytokines and lung epithelial injury markers, as assessed by plasma and alveolar soluble receptors for advanced glycation end products (sRAGE)²⁸. As sRAGE is now considered as a marker of alveolar type 1 cell injury and a key mediator of alveolar inflammation, these results could suggest some beneficial effects of sevoflurane on the lung alveolar epithelial injury^21,29,30.

The use of halogenated agents for inhaled ICU sedation has long required operating room anesthesia ventilators and gas vaporizers to be deployed in the ICU. Since then, anesthetic reflectors suitable for the use with modern critical care ventilators have been developed for specific use in the ICU³¹. These devices feature modified heat and moisture exchanging filters inserted between the Y-piece of the respiratory circuit and the endotracheal tube. They allow the administration of halogenated agents, with isoflurane and sevoflurane being the most frequently used, and they consist of a porous polypropylene evaporator rod, into which a liquid agent, delivered by a specific syringe pump, is released. The halogenated agent is absorbed during expiration by a reflecting medium contained in the device and it is released during the next inspiration, allowing recirculation of approximately 90% of the expired halogenated agent^31,32. Recently, a miniaturized version of the device was developed with an instrumental dead space of 50 mL, making it even more suitable for use during ultra-protective ventilation in ARDS patients, with tidal volumes that could be as low as 200 mL³¹. Such a miniaturized device has never been studied in an experimental piglet model of ARDS.

Because previous research supports the promising roles of halogenated volatiles in lung alveolar inflammation and injury during ARDS, we designed an experimental animal model to achieve a translational understanding of the mechanisms of the effects of halogenated agents on lung injury and repair^33,34,35. In this study, we developed a model of hydrochloric acid (HCl)-induced ARDS in piglets in whom inhaled sedation can be delivered using the miniaturized version of the anesthetic conserving device, an ICU-type device. This large animal model of ARDS could be used to further our understanding of the potential lung-protective effects of inhaled halogenated agents.

Protocol

The study protocol was approved by the Animal Ethics Committee of the French Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (approval number 01505.03) before being registered at preclinicaltrials.eu (Pre-clinical registry identifier PCTE0000129). All procedures were performed in the Centre International de Chirurgie Endoscopique, Université Clermont Auvergne, Clermont-Ferrand, France, in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines³⁶.

1. Animal preparation and anesthesia

1. Piglet mode
   1. Ensure that the experimental protocol is consistent with guidelines for animal experiments, including the 3R principles (replacement, reduction, and refinement) and national/international regulations.
   2. Obtain approvals from the ethics committee for care and use of experimental animals at the relevant institution before starting the protocol.
   3. Use a male white Landrace piglet (2–4 months old; weighing 10–15 kg).
   4. Place the piglet in the supine position after premedication using intramuscular azaperone (described in 1.2.2).
2. Anesthesia induction
   1. Restrict animals from having food for overnight while allowing free access to water.
   2. Administer anxiolytic premedication to the piglet using intramuscular azaperone (2 mg.kg^-1) behind the ear.
   3. Apply a finger pressure on the soft tissues of the auricular base of the piglet to identify the medial and lateral auricular vein.
   4. Insert a peripheral intravenous 22 G catheter in the medial or lateral auricular vein of the piglet. Follow with the catheter at a shallow angle of 45˚ through the skin and advance until blood appears through the catheter.
   5. Induce general anesthesia with intravenous propofol (3 mg.kg^-1) and sufentanil (0.3 µg.kg^-1)³⁷. Check the depth of the anesthesia by lack of response to pedal reflex.
3. Tracheal intubation^38,39
   1. Prepare the laryngoscope using a size 4 straight Miller laryngoscope blade.
   2. Pass the laryngoscope into the pharyngeal cavity and depress the tongue with the laryngoscope blade, making the epiglottis visible.
   3. Visualize the larynx opening of the piglet prior to orotracheal intubation.
   4. Insert a 6 mm internal diameter cuffed endotracheal tube.
   5. Inflate the endotracheal tube cuff to reach a cuff pressure around 20–30 cmH₂O.
   6. Fix the endotracheal tube to the piglet’s nose with micropore surgical tape.
   7. Connect to the ventilator and initiate mechanical ventilation following the settings described in section 3.
4. Sedation maintenance
   1. Maintain anesthesia with continuous intravenous infusion of propofol (5 mg.kg^-1.h^-1) before acid-induced lung injury. The infusion of propofol will be stopped when halogenated agents are started.
   2. Add a continuous intravenous infusion of remifentanil (10–20 μg.kg⁻¹.h⁻¹ = 0.15–0.33 μg.kg⁻¹.min⁻¹) for pain management.
   3. Add continuous intravenous infusion of cisatracurium (0.2 mg.kg^-1.h^-1) for a neuromuscular blockade.
   4. Keep the body temperature of the piglet at approximately 38 °C using warm blankets.
   5. Monitor electrocardiogram activity, the peripheral oxygen saturation (SpO₂), and arterial pressure continuously using an external monitor.
5. Surgery
   1. Insert central venous access using a surgical exposure of the right internal jugular vein and the Seldinger method to insert a 3-lumen catheter (7 French, 16 cm).
      1. Make a cutaneous midline incision on the ventral aspect of the neck, 2 cm lateral from the trachea. Use surgical forceps to dissect the tissues.
      2. Localize the internal jugular vein (approximately 1–2 cm deep, lateral to the internal carotid artery) and, using the needle (18 G, 6.35 cm), make a puncture with a craniocaudal direction orientation.
      3. With the hand, insert the “J” guidewire (0.81 mm diameter, 60 cm) through the needle. Gently remove the needle and quickly insert a venous catheter with three lines into the internal jugular vein along the “J” guidewire. Remove the “J” guidewire while maintaining the venous catheter in place.
      4. Aspirate blood through each line of the venous catheter to remove the air from the different lines and flush with 5 mL of saline solution (0.9% NaCl) to rinse the three lines.
      5. Suture the skin with a 3.0 non-absorbable suture thread following the continuous Lembert pattern and fix the catheter to the skin with a single stitch and triple knots on each lateral perforation of the central venous catheter.
   2. Insert an arterial line via surgical exposure of the right femoral artery and use the Seldinger method to insert the thermodilution catheter (3–5 French, 20 cm).
      1. Place the right forelimb of the piglet in extension.
      2. Make a cutaneous incision on the right groin area of the piglet. Use surgical forceps to dissect the subcutaneous and muscular tissues.
      3. Localize the right femoral artery by palpating the femoral pulse (approximately 3–4 cm deep) and, using the needle (19 G, 54 mm), make a puncture with a caudocranial direction orientation.
      4. Insert the “J” guidewire through the needle. Gently remove the needle and quickly insert an arterial catheter into the femoral artery up along the guidewire. Remove the guidewire while maintaining the catheter in place.
      5. Remove the air from the arterial catheter and flush with saline solution to rinse the line.
      6. Suture the skin with a 3.0 non-absorbable suture thread following the continuous Lembert pattern and fix the catheter to the skin with a single stitch and triple knots on each lateral perforation of the arterial catheter.
      7. Plug the catheter on an arterial line tubing to allow retrieval of serial blood samples and continuous hemodynamic monitoring (arterial pressure, cardiac index, and extravascular lung water, as indexed to body weight) with a pulse contour cardiac output monitor device.

2. Acid-induced acute lung injury

CAUTION: Use gloves and glasses during this step to avoid any risk of contact of the acid with the skin or the eyes)

1. Make 100 mL of HCl at 0.05 M and pH 1.4.
2. Using the anatomical landmark of the last segment of the sternum, measure the distance between the tip of the endotracheal tube and the carina of the piglet.
3. Mark this distance with a black pen on a Ch14 suction catheter.
4. Insert the suction catheter through the endotracheal tube up to the black landmark.
5. Gently instill 4 mL.kg^-1 (bodyweight) of acid through the suction catheter for over 3 min.
6. Remove the suction catheter.

3. Mechanical ventilation

1. Use volume-controlled ventilation on an intensive care ventilator.
2. Use a tidal volume of 6 mL.kg^-1, a positive end-expiratory pressure (PEEP) of 5 cmH₂O, and an inspired oxygen fraction (FiO₂) of 40%.
3. Adjust the respiratory rate to maintain the end-tidal carbon dioxide between 35 and 45 mmHg.
   NOTE: Based on previous studies^37,40,41, lung injury is considered established when the arterial oxygen tension (PaO₂)-to-FiO₂ ratio decreases to 25% from the baseline, approximately 1 h after airway HCl instillation.

4. Halogenated anesthetics

NOTE: Start sedation using halogenated anesthetics (sevoflurane or isoflurane) once acid-induced lung injury is achieved. The intravenous sedation using propofol should then be interrupted.

1. Filling the syringe (Figure 1A): Attach the filling adapter provided by the manufacturer to the 250 mL bottle of the halogenated agent and a 60 mL syringe to the filling adapter. Turn the bottle upside down and fill the syringe by pushing and pulling the plunger. Turn the bottle upright and remove the syringe.
2. Scavenging (Figure 1B)
   1. Place the charcoal filter, used to remove halogenated hydrocarbon anesthetic gases, close to the ventilator.
   2. Remove the protective cap from the charcoal filter.
   3. Connect the charcoal filter to the expiratory valve of the ventilator with a flex tube.
3. Use the anesthetic conserving device (device used for inhaled ICU sedation) (Figure 1C) as described below.
   1. Connect the ionomer membrane dryer line to the gas sampling port of the anesthetic conserving device.
   2. Connect one side of the gas sampling line to the ionomer membrane dryer line.
   3. Connect the other side of the gas sampling line to the gas analyzer.
   4. Insert the anesthetic conserving device between the Y-piece of the respiratory circuit and the endotracheal tube.
   5. Ensure that the anesthetic conserving device has the black side up and is sloped down toward the piglet.
4. Deliver inhaled sedation through the anesthetic conserving device (Figure 2).
   1. Place the specific syringe in the syringe pump.
   2. Connect the anesthetic agent line to the syringe.
   3. Prime the agent line with a bolus of 1.5 mL of the halogenated agent.
   4. Adapt the initial pump rate in mL.h^-1 (initial syringe pump rate settings of isoflurane and sevoflurane are 3 and 5 mL/h, respectively) to the targeted expired sevoflurane fraction (F_Esevo) or the expired isoflurane fraction (F_Eiso) value, as displayed on the gas analyzer.
   5. Ensure that the gas analyzer displays a F_Esevo %–F_Eiso % or equivalent minimal alveolar concentration value greater than zero. If necessary, give an additional bolus of 0.3 mL of the halogenated agent.
   6. Adapt the syringe pump rate necessary to reach a certain concentration depending on the minute volume and the targeted concentration, with rates of 2–7 mL.h^-1 and 4–10 mL.h^-1 being, in general, associated with expired fractions of 0.2%–0.7% and 0.5%–1.4% for isoflurane⁴² and sevoflurane^28,43, respectively.
   7. During the experiment, continue administration of the halogenated agents with F_Esevo and F_Eiso targets of 0.8–1.1 and 0.5–0.8, respectively.

5. Measurements

1. Monitoring
   1. Collect different parameters as measured by the external monitor: heart rate, blood pressure, and peripheral oxygen saturation.
   2. Record parameters as measured by the ventilator: tidal volume, respiratory rate, set PEEP, auto-PEEP (by applying an expiratory hold maneuver of 5 s on the ventilator), compliance of the respiratory system, airway resistance, inspiratory plateau pressure (by applying an inspiratory hold maneuver of 2 s on the ventilator), peak inspiratory pressure, and driving pressure.
   3. Calculate the lung functional residual capacity using the Nitrogen Wash In/Wash Out method if integrated in the ventilator.
   4. Use the thermal indicator previously inserted in the femoral artery to measure the extravascular water volume of the lungs, cardiac index, and systemic vascular resistance.
2. Undiluted pulmonary edema fluid sampling to measure the net AFC rate.
   1. Insert a soft 14 Fr suction catheter into a wedged position in the distal bronchus through the endotracheal tube.
   2. Sample edema fluid into a suction trap by applying gentle suction.
   3. Centrifuge all samples at 240 x g at 4 °C for 10 min in a refrigerated centrifuge.
   4. Collect the supernatants.
      NOTE: Total protein concentration in undiluted pulmonary edema fluid is measured with a colorimetric method. Because the rate of clearance of edema fluid from the alveolar space is much faster than the rate of protein removal, the net AFC rate was calculated as Percent AFC = 100 × [1 - (initial edema protein/final edema total protein)] and thereafter was reported as %/h³⁷. Undiluted pulmonary edema fluid samples are collected from the animals at baseline and 4 h later, as previously described^{34,44,45,46,47,48,49}.
3. Mini bronchoalveolar lavage sampling.
   1. Insert a soft 14 Fr suction catheter into a wedged position in a distal bronchus through the endotracheal tube.
   2. Instill 50 mL of a 0.9% sodium chloride solution into the suction catheter.
   3. Promptly sample the fluid into a suction trap.
   4. Collect the mini bronchoalveolar lavage.
      NOTE: Total protein concentration in mini BAL is measured with a colorimetric method and, for example, the levels of proinflammatory cytokines, such as TNF-α, IL-6, IL-1β, and IL-18, are measured using a multiplex immunoassay method. Samples are collected 4 h after the acid-induced lung injury.
4. Blood gas analysis
   1. Collect arterial blood gases through the arterial line in a 3 mL BD Preset syringe with BD Luer-Lok tip at baseline. Immediately measure PaO₂/FiO₂, PaCO₂, pH, serum lactate, and serum creatinine using a point-of-care blood gas analyzer.
   2. Repeat this step every hour for 4 h after acid instillation.
5. Lung sampling
   1. Sacrifice the piglet with an intravenous injection of pentobarbital (150 mg.kg^-1) at the end of the experiment (4 h after acid-induced lung injury).
   2. Dissect and remove the whole lungs. Fix with alcohol acetified formalin.
   3. Embed in paraffin and slice at a 10 μm thickness.
   4. Stain with hematoxylin and eosin.
      NOTE: Histological evidence of lung injury can be assessed using a standardized histology injury score⁵⁰.

Results

For this experiment, 25 piglets were anesthetized and divided in two groups: 12 piglets in the untreated group (SHAM group) and 13 piglets in the acid-injured group (HCl group). No piglet died before the end of the experiment. A two-way repeated-measures analysis of variance (RM-ANOVA) indicated a significant time by group interaction (P < 10⁻⁴) with a detrimental effect of HCl-induced ARDS on PaO₂/FiO₂, compared to sham animals without ARDS (Figure 3

Discussion

This article describes a reproducible experimental model of ARDS induced by the intratracheal instillation of HCl in piglets to investigate the lung-protective effects of halogenated volatiles, such as sevoflurane or isoflurane, delivered using an anesthetic conserving device.

The primary goal of this study was to develop an experimental model of ARDS in which volatile agents could be delivered by an anesthetic conserving device, such as those used in ICU patients. Although some effects of hal...

Materials

Name	Company	Catalog Number	Comments
Tracheal intubation
Endotracheal tube 6-mm	Covidien	18860
Animal preparation
Central venous catheter 3-lumens catheter (7 French - 16 cm)	Arrow	CV-12703
Pulse contour cardiac output monitor PiCCO catheter (3-5 French - 20 cm)	Getinge Pulsion Medical System	catheter
Warm blankets WarmTouch5300	MedTronic	5300
Monitoring
External monitor IntelliVue MP40	Phillips	MNT 142
Point-of-care blood gas analyzer Epoc® Blood Analysis System	Siemens	20093
Pulse contour cardiac output monitor PiCCO Device PulsioFlex Monitor	Getinge Pulsion Medical System	PulsioFlex
Mechanical ventilation
Ventilator Engström Carestation	General Electrics	Engström
Halogenated anesthetics
Anaconda Syringe	SedanaMedical	26022
Anesthetic conserving device AnaConDa-S	SedanaMedical	26050
Charcoal filter FlurAbsorb	SedanaMedical	26096
Filling Adaptaters	SedanaMedical	26042
Ionomer membrane dryer line Nafion	SedanaMedical	26053
Products
Propofol	Mylan	66617123
Isoflurane	Virbac	QN01AB06
Pentobarbital	PanPharma	68942457
Sevoflurane	Abbvie	N01AB08
Sufentanil	Mylan	62404996