We thank all colleagues and professionals who contributed to and supported the construction of this ex vivo porcine lung model protocol.
This study had no funding sources.

The protocol was approved by the Animal Research Ethics Committee of our Institution (protocol no. 1610/2021).
1. Anesthesia and animal preparation
<ol>
	<li>Initially, place the animal on a scale and check the weight to adjust the medications and sedation necessary for the procedure.</li>
	<li>Administer ketamine 5 mg/kg and midazolam 0.25 mg/kg intramuscularly.</li>
	<li>Puncture the marginal ear vein with a 20 G venous catheter and administer intravenous propofol (5 mg/kg) for anesthesia induction.</li>
	<li>Administer 3 mL of heparin intravenously into the access to the marginal ear vein to assist in the cardiopulmonary extraction and perfusion.</li>
	<li>After anesthesia, perform orotracheal intubation with a 6.5 mm orotracheal cannula (OTC) and fix the OTC with adhesive tape, leaving it firmly fixed to avoid displacement during the procedure. 
	​NOTE: The depth of sedation is checked by monitoring hemodynamic parameters and using a gas analyzer, such as mean arterial pressure, heart rate, and inspired/expired isoflurane concentration.</li>
</ol>
2. Intraoperative mechanical ventilation
<ol>
	<li>Connect the animal via the OTC to MV, maintaining sedation with 1.5% isoflurane at 50% of the inspired fraction of oxygen (FiO2) and fentanyl 10 mcg/kg bolus + 10 mcg/kg/h continuous infusion.
	<ol>
		<li>Tap on the mechanical ventilator screen and select the volume-controlled ventilation (VCV) mode, select the tidal volume (TV) button, and turn the scroll wheel until the tidal volume value corresponds to 8 mL/kg.</li>
		<li>Tap on the mechanical ventilator screen. Select the FiO2 and turn the scroll wheel until the value of 50% is reached.</li>
		<li>Tap on the mechanical ventilator screen and select the respiratory rate (RR). Turn the wheel until it reaches the ideal value to maintain an end-expired CO2 of 35-45 mmHg measured by capnography coupled to the mechanical ventilator. 
		​NOTE: The depth of sedation is checked by monitoring hemodynamic parameters and using a gas analyzer such as mean arterial pressure, heart rate, and inspired/expired isoflurane concentration.</li>
	</ol>
	</li>
</ol>
3. Tissue dissection and OTC exchange
<ol>
	<li>Make a medial sternal incision from 2 cm above the manubrium to 2 cm below the xiphoid process of the sternum to access the thoracic cavity. Position the rib retractors, expanding the field of view during the procedure.</li>
	<li>Use a scalpel to make a horizontal tracheal incision at the height of the cricoid cartilage (just at the first tracheal rings) wide enough to introduce a new tracheal cannula.</li>
	<li>Deflate the OTC cuff that is inside the airway and pull slowly to remove it. Meanwhile, insert the new OTC into the incision made in the trachea after removing the old OTC. Leakage may occur due to the deflated cuff, ceasing when repositioning the new OTC.</li>
	<li>Inflate the newly inserted tracheal tube cuff by connecting a 20 mL syringe to the pilot balloon. The syringe delivers air under pressure and inflates the pilot balloon and cuff. Once the cuff inflates, remove the syringe.</li>
	<li>Tie the new tracheal cannula directly to the trachea with 2-0 polyester to prevent leakage and movement while placing the lung in the plexiglass ventilation box.</li>
	<li>With the scalpel, dissect the tissues to remove the cardiopulmonary organs from the thorax.</li>
</ol>
4. Animal euthanasia
<ol>
	<li>Increase the isoflurane concentration to 5% and administer 10 mL of 19.1% potassium chloride. Subsequently, check the absence of vital signs. 
	​NOTE: This procedure was performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.</li>
</ol>
5. Cardiopulmonary extraction
<ol>
	<li>After euthanasia, dissect the respiratory ligament to remove the lungs.</li>
	<li>After the tissue dissection, clamp the OTC with the appropriate Kelly forceps during the end of inspiration, keeping the lungs inflated.</li>
	<li>Disconnect the OTC from the mechanical ventilator, but keep it clamped.</li>
	<li>Section the aortic artery, position the aspirator inside the thoracic cavity to remove the extravasated blood, maintain visualization of the cavity while finishing the dissections, and free the organs to be removed from the thoracic cavity. 
	NOTE: The inferior pulmonary ligament should be carefully released to avoid pulmonary laceration.</li>
	<li>Remove the heart and lung from the ribcage with the OTC clamped, without separating them, and place them on a tray.</li>
</ol>
6. Cardiopulmonary preparation
<ol>
	<li>With the lung on a tray, cannulate the pulmonary artery with a large-bore single-lumen catheter and connect it to the infusion set to continuously administer 2,000 mL of cold 0.9% saline solution (SS) or until clear liquid flows from the aorta. 
	NOTE: SS should be administered at a normal rate, avoid squeezing the intravenous (IV) bag.</li>
	<li>After clearing the flow, suture the aortic artery with 2-0 polyester and administer another 100 mL of 0.9% SS. Close the single-lumen catheter outlet as the liquid will remain inside until the end of the experiment.</li>
	<li>Unclamp the OTC, note that the lungs will deflate and remain closed, ready to receive MV and the ARM.</li>
</ol>
7. MV inside an acrylic box
<ol>
	<li>After preparation, open the acrylic box and position the lungs vertically inside the box. Pass the OTC through the hole in the lid and connect the tracheal cannula to the mechanical ventilator. 
	NOTE: Make sure the tracheal cannula is firmly secured in the trachea.</li>
	<li>Select the Start Ventilation button.
	<ol>
		<li>Tap on the mechanical ventilator screen and select the mechanical ventilator for VCV.</li>
		<li>Tap on the VCV mode settings screen, and select the TV button, turn the wheel until it reaches the value of 6 mL/kg. Do the same to adjust PEEP to 5 cm H2O, FiO2 to 21%, RR to 15 breaths per minute, and inspiratory pause time to 10%.</li>
	</ol>
	</li>
</ol>
8. ARM
<ol>
	<li>To start the recruitment, increase PEEP from 5 cm H2O to 6 cm H2O and then increase it in step-by-step increments of 2 cm H2O until reaching 14 cm H2O.PEEP is increased using the on-screen button below the PEEP value displayed on the screen. Turn the wheel to increase the value.
	<ol>
		<li>For each PEEP, write down the peak pressure, plateau pressure, dynamic compliance, and airway resistance values displayed on the mechanical ventilator screen. Write down the driving pressure, which is the plateau pressure value minus the PEEP value adjusted at that time.</li>
	</ol>
	</li>
	<li>After reaching 14 cm H2O, reduce PEEP in step-by-step decrements of 2 cm H2O until reaching 6 cm H2O, then reduce it to 5 cm H2O. PEEP is reduced using the on-screen button below the PEEP value displayed on the screen. Turn the wheel to decrease the value.
	<ol>
		<li>For each PEEP, write down the peak pressure, plateau pressure, dynamic compliance, and airway resistance values displayed on the mechanical ventilator screen. Write down the driving pressure, which is the plateau pressure value minus the PEEP value adjusted at that time. 
		​NOTE: Maintain each PEEP value for 10 min during the increment and for 5 min at each step during the decrement.</li>
	</ol>
	</li>
</ol>
9. Cardiopulmonary maintenance
<ol>
	<li>At the end of the recruitment stage, gently clamp the tracheal cannula with the clamp during inspiration, keeping the lungs inflated. Open the acrylic box.</li>
	<li>Remove the lungs from the acrylic box and carefully place them in a glass container. 
	​NOTE: Make sure the tracheal cannula is firmly secured in the trachea.</li>
	<li>Pour 500 mL of 0.9% SS.</li>
	<li>Store it in the refrigerator in a plastic-wrapped glass container at a temperature of 2 to 8 °C for 24 h.</li>
	<li>Repeat the steps 7, 8, and 9 for five consecutive days.</li>
</ol>
<img alt="Flowchart of cardiopulmonary block preparation and assessment, including lung mechanics evaluation." class="xfigimg" src="/files/ftp_upload/64850/64850fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Study flowchart. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

The described protocol is useful for producing an ex vivo porcine lung model under positive-pressure MV. It can be used for studying and teaching lung mechanics through visual feedback from the lungs during recruitment and analysis of the curves and values projected on the device screen. To achieve this result, pilot studies are needed to understand the behavior of the lungs outside the rib cage and to identify the need for adaptations.
We identified that the critical point was the formation of bubbles, fistulas, and lesions in the pleura that were visualized when connecting the mechanical ventilator, with a difference between inspired and expired TV and changes in the volume curve. Thus, one of the first protocol modifications was to use a wide surgical opening of the thorax, with diaphragm incision at the beginning of the procedure during the dissection of the cardiopulmonary organs, which can improve visualization of the structures and help the careful release of the inferior pulmonary ligament, maintaining lung integrity. Furthermore, manual inflation of the pilot lungs after the structures were dissected showed that this inflation exceeds the pressure limits and contributes to the formation of blisters and fistulas. Some studies using ex vivo lungs presented the possibility of using fibrin glue for leaks, with positive results; although we did not use this approach in the study, it could be an alternative to improve the model26,27. Another relevant point is that the lungs were removed and completely deflated in the pilot study, keeping them totally collapsed from organ preparation to MV initiation, which made it difficult to open the lungs to MV and increased the possibility of fistula formation. Hence, we started to clamp the OTC and keep the lungs inflated during the dissection until SS was administered. Afterward, the OTC was released, deflated, and connected the lungs to the mechanical ventilator to start the ARM, and an analysis of lung mechanics was performed to demonstrate the pulmonary hysteresis curve. This did not compromise lung recruitment or the analysis of lung mechanics because anesthetized patients have atelectasis and reduced lung compliance even during MV28,29,30,31.
In the pilot study, an initial PEEP of 5 cm H2O was used and increased in 5 cm H2O increments up to 25 cm H2O32,33. However, the peak and plateau pressures reached values greater than 40 and 30 cm H2O, respectively, with fistula formation. Thus, a gradual increase in 2 cm H2O increments was performed to better analyze the behavior of pressures over time and to understand PEEP limits in our ex vivo lung model. There was no difference in mortality between sustained and incremental inflation, but incremental inflation is the most used and can facilitate the stepwise analysis of lung mechanics34. As for the use of negative pressure20,21, the model was tested only under positive pressure because patients on MV are subjected to positive pressure. We do not rule out the use of negative pressure in the future, but it would require acrylic case changes.
The literature presents some models produced with a test lung, pistons, and an ex vivo model13,14 that were placed in hermetically sealed boxes that simulated the ribcage. Our model was placed in a conventional acrylic box, which, despite reducing the possibility of applying negative pressure, can facilitate the production of the model. Another model produced for preclinical studies18 is similar to ours, but the lungs were positioned horizontally while ours were maintained vertically, receiving the action of gravity without the support of the organs and ribcage. These lungs were used during experiments within 48 hours after euthanasia18,19,20,21,35. Our model was used for a total of 120 h, being kept at a temperature of 2-8 &#176;C during the 24 h of the experiment, showing the positive results described in the representative results section.
The gap in teaching and training was not addressed at this first moment, but the model is effective for analyzing lung mechanics and can be used as a tool for research and teaching. In addition, we did not aim to study perfusion solutions, but in the same way that we infused SS in step 6.1, perfusion and preservation solutions can be used, opening new possibilities for studies with the same model presented.
This technique has some limitations: 1) knowledge of animal anatomy to ensure that the lungs are removed properly; 2) the model was not evaluated beyond five days; 3) the model appears to be appropriate for teaching ventilation but has not been tested in a teaching context; 4) it is an animal model, so it is important to consider its applicability limitations in humans.

Mechanical ventilation (MV) is widely used in intensive care units (ICUs) and surgical centers. Its monitoring is essential to help recognize asynchronies and prevent injuries for all patients, especially when the patient has serious lung injuries1,2,3,4,5,6. Monitoring respiratory mechanics can also contribute to the clinical understanding of the disease progression and therapeutic applications, such as the use of positive end-expiratory pressure (PEEP) or the alveolar recruitment maneuver (ARM). However, the use of these techniques requires a proficient understanding of curves and basic lung mechanics3,4.
Students, residents, and medical professionals feel insecure about MV management, from turning on the ventilator and initial adjustments to monitoring plateau and driving pressures, and this insecurity is associated with a lack of knowledge and adequate prior training7,8,9,10. We observed that professionals who participated in simulations and used a lung model reported greater confidence, understanding of the parameters, and understanding of the components of lung mechanics8,11,12.
Models for studying and training MV with test lungs, bellows, and pistons can simulate different pressures and volumes, as well as different lung mechanics conditions13,14,15. Computational and software models also contribute to the study of cardiopulmonary interaction by generating simulations that can be used to teach the principles of MV11 to health professionals16,17.
While computational models may present difficulties in representing pulmonary hysteresis16, models with test lung and bellows13,14,15can produce pressure-volume curves similar to the physiological curve and demonstrate pulmonary dynamics. As an advantage, the ex vivo porcine lung presents similar anatomy to humans18, also producing MV curves, pulmonary hysteresis, and providing visual feedback of the lungs inside the acrylic box during the lung mechanics analysis. Visual models are important and can help understand difficult-to-imagine components and concepts. Thus, ex vivo lung models represent a practical way of teaching.
Studies with ex vivo porcine lungs, such as those on MV with positive and negative pressure19,20,21, analysis of aerosol distribution22,23, pediatric simulations24, and lung perfusion25 can improve the knowledge on MV. Recent studies analyzing models in positive and negative pressure have shown that positive-pressure ventilation can lead to abrupt recruitment with greater local deformation, greater distension, hysteresis curve differences, and possible tissue lesions compared to negative pressure pressure19,20,21. Nevertheless, positive-pressure models are necessary because patients are under positive pressure during MV pressure19,20,21. The development of a lung model for preclinical studies opens possibilities for new research and applications, including MV teaching and training.
Here, we present an ex vivo porcine lung model for studying and training purposes. Our primary objective is to describe the steps for the generation of this ex vivo porcine lung model under positive-pressure MV. It can be used in the future to study and teach lung mechanics.

<table><tbody><tr><td>0.9% Saline solution</td><td></td><td></td><td>2500ml</td></tr><tr><td>Anesthesia machine - Primus</td><td>Drager</td><td>REF 8603800-18</td><td>Anesthesia work station used in the procedure</td></tr><tr><td>Aspirator</td><td></td><td></td><td>For blood aspiration from thorax</td></tr><tr><td>Bedside Monitor - Life Scope</td><td>Nihon Kohden</td><td>BSM-7363</td><td>Multiparameter monitor used during the procedure</td></tr><tr><td>Bonney Tissue Forceps</td><td></td><td></td><td>Any tissue forceps is suitable</td></tr><tr><td>Disposable scalper, #23</td><td></td><td></td><td>Any scalper is suitable</td></tr><tr><td>Disposable syringe needles, 18G x 1 1/2&quot;, 23G x 1&quot;</td><td>BD</td><td>302814</td><td>Widely available</td></tr><tr><td>Disposable syringes, 10ml</td><td></td><td></td><td>Widely available</td></tr><tr><td>Electrosurgical unit - SS-501</td><td>WEM</td><td></td><td>For cutting and coagulation during thorax incision</td></tr><tr><td>Fentanyl</td><td></td><td></td><td>10 mcg/kg bolus + 10 mcg/kg/hour continuous infusion</td></tr><tr><td>Finochietto retractor</td><td></td><td></td><td>Any finochietto retractor is suitable</td></tr><tr><td>heparin</td><td></td><td></td><td>3ml</td></tr><tr><td>Infusion set</td><td></td><td></td><td>Any infusion set is suitable</td></tr><tr><td>Isoflurane</td><td></td><td></td><td>1.5%</td></tr><tr><td>Kelly Forceps Curved</td><td></td><td></td><td>Any kelly forceps is suitable</td></tr><tr><td>Ketamine</td><td></td><td></td><td>5mg/kg</td></tr><tr><td>Lactated Ringer solution</td><td></td><td></td><td>500ml</td></tr><tr><td>Mechanical ventilator - Servo I</td><td>Maquet</td><td>REF 6449701</td><td>Mechanical ventilator used in the procedure</td></tr><tr><td>Metzenbaum Scissor (Straight and curved)</td><td></td><td></td><td>Any metzenbaum scissor is suitable</td></tr><tr><td>Midazolam</td><td></td><td></td><td>0.25mg/kg</td></tr><tr><td>Orotracheal intubation cannula, #6.5</td><td>Rusch</td><td>112282</td><td>Widely available</td></tr><tr><td>Plexiglass</td><td></td><td></td><td>Custom made plexiglass box: 30x45x60cm</td></tr><tr><td>Polyester suture, 2-0</td><td></td><td></td><td>Widely available</td></tr><tr><td>Potassium choride</td><td></td><td></td><td>10 ml, 19.1% potassium chloride.</td></tr><tr><td>propofol</td><td></td><td></td><td>5mg/kg</td></tr><tr><td>Three way stopcock</td><td></td><td></td><td>Widely available</td></tr><tr><td>Venous catheter, G20 x 1&quot;</td><td>BD</td><td>38183314</td><td>Widely available</td></tr></tbody></table>

ex vivo porcine experimental model for studying and teaching lung mechanics

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel insecure and lack knowledge because of inadequate training and teaching methods. Therefore, the objective of this article is to outline the steps involved in generating an ex vivo porcine lung model to be used in the future, to study and teach lung mechanics. To generate the model, five porcine lungs were carefully removed from the thorax following the guidelines of the Animal Research Ethics Committee with adequate care and were connected to the mechanical ventilator through a tracheal cannula. These lungs were then subjected to the alveolar recruitment maneuver. Respiratory mechanics parameters were recorded, and video cameras were used to obtain videos of the lungs during this process. This process was repeated for five consecutive days. When not used, the lungs were kept refrigerated. The model showed different lung mechanics after the alveolar recruitment maneuver every day; not being influenced by the days, only by the maneuver. Therefore, we conclude that the ex vivo lung model can provide a better understanding of lung mechanics and its effects, and even of the alveolar recruitment maneuver through visual feedback during all stages of the process.

We used five female pigs weighing between 23.4-26.9 kg and followed the described protocol for cardiopulmonary extraction and lung mechanics analysis. Our intention is that the model is useful for the study of lung mechanics by analyzing peak pressure, plateau pressure, resistance, driving pressure, and dynamic compliance variables collected directly from the mechanical ventilator screen. The model flowchart is shown in Figure 1.
The lungs were analyzed for five consecutive days, repeating the entire process described in items 7.2, 8.1, 8.2, 9.1, 9.2, and 9.3 of the protocol. We tried to show how lung variables behaved pre- and post-recruitment and to verify the durability of the ex vivo pulmonary model in the established period.
Significant differences (p &lt; 0.05) were observed for all variables between pre- and post-ARM. The peak pressure, plateau pressure (Figure 2), and driving pressure (Figure 3) decreased after the maneuver (p = 0.0005), while dynamic compliance (p = 0.0007) increased (Figure 4), demonstrating open collapsed alveoli and lung area gain. Resistance (Figure 5) also increased after recruitment (p = 0.0348). None of the variables were significantly influenced by the day.
Based on these results, we showed that the model is effective in demonstrating visual lung mechanics changes through the ARM (Figure 6) and in studying and teaching lung mechanics (Figure 7). In addition, we showed that the model can be used for at least five consecutive days. As we did not evaluate the model beyond this period, we cannot confirm the final durability of the lung model.
<img alt="Peak and plateau pressure trends, graph, data analysis, respiratory study, cmH₂O, 5-day measurement." class="xfigimg" src="/files/ftp_upload/64850/64850fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Pressures. (A) Peak pressure. The pre-ARM Ppeak ranged from 21 ± 3.2 to 23 ± 2.3 cmH2O, while the post-ARM Ppeak ranged between 9 ± 0.6 and 12.6 ± 1.4 cmH2O in the five lungs. The two-way ANOVA statistical analysis was used to calculate the p-value of 0.0005, which was considered significant. (B) Plateau pressure. The pre-ARM Pplateau ranged from 21 ± 3.2 to 22 ± 2.3 cmH2O, while the post-ARM Pplateau ranged between 8.8 ± 0.4 and 11.6 ± 1.6 cmH2O in the five lungs. The two-way ANOVA statistical analysis was used to calculate the p-value of 0.0005, which was considered significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Driving pressure vs. days chart; p=0.0005; clinical data analysis; error bars indicate variance." class="xfigimg" src="/files/ftp_upload/64850/64850fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Driving pressure. The pre-ARM Driving pressure ranged from 16 ± 3.2 to 17 ± 2.3 cmH2O, while the post-ARM Driving pressure ranged between 3.8 ± 0.4 and 6.6 ± 1.6 cmH2O in the five lungs. The two-way ANOVA statistical analysis was used to calculate the p-value of 0.0005, which was considered significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dynamic compliance graph showing mL/cmH2O over 5 days with statistical significance p=0.0007." class="xfigimg" src="/files/ftp_upload/64850/64850fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Dynamic compliance. The pre-ARM Dynamic compliance ranged from 9.1 ± 1.2 to 10.2 ± 2.6 mL/cmH2O, while the post-ARM Dynamic compliance ranged between 23.6 ± 3.5 and 43.8 ± 11.3 mL/cmH2O in the five lungs. The two-way ANOVA statistical analysis was used to calculate the p-value of 0.0007, which was considered significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Resistance analysis chart; cmH2O/L/s vs. days; line graph comparing pre/post ARM results." class="xfigimg" src="/files/ftp_upload/64850/64850fig05v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Resistance. The pre-ARM Resistance ranged from 1.4 ± 1.0 to 7 ± 3.2 cmH2O/L/seg, while the post-ARM Resistance ranged between 2.4 ± 0.4 and 6.6 ± 5.1 cmH2O/L/seg in the five lungs. The two-way ANOVA statistical analysis was used to calculate the p-value of 0.0348, which was considered significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lung inflation sequence; six stages; biological experiment demonstrating respiratory mechanics." class="xfigimg" src="/files/ftp_upload/64850/64850fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Lung model. (A) Lung with PEEP of 5 cm. (B) Lung with PEEP of 6 cm. (C) Lung with PEEP of 8 cm. (D) Lung with PEEP of 10 cm. (E) Lung with PEEP of 12 cm. (F) Lung with PEEP of 14 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Ventilator monitor displaying respiratory waveforms; volume, pressure curves; patient status data." class="xfigimg" src="/files/ftp_upload/64850/64850fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7. Mechanical ventilation charts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64850/64850fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics at JoVE.com

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

We present an ex vivo pig lung model for the demonstration of pulmonary mechanics and alveolar recruitment maneuvers for teaching purposes. The lungs can be used for more than one day (up to five days) with minimal changes in pulmonary mechanics variables.

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel ...

ex-vivo-porcine-experimental-model-for-studying-teaching-lung

Nanyang Technological University

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JoVE Journal

Medicine

1.3K Views.  Universidade de São Paulo. Mechanische beademing wordt veel gebruikt bij respiratoire insufficiëntie, gedeeltelijk of volledig ter vervanging van spontane beademing. Het beheer ervan vereist voorkennis en expertise. Sommige onderzoeken tonen echter aan dat professionals zich onzeker voelen in het beheer ervan.Elke claim, gebrek aan training of voorkennis. De experimentele modellen komen om de concepten van mechanische ventilatie en visualisatie van longmechanica met visuele feedback te vergemakkelijken. Aanvank...

Video: Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem of a low number of lungs considered viable for the donation process. In recent decades, however, this scenario has been positively changed, given the development of ex vivo lung perfusion (EVLP) as a strategy for evaluating and reconditioning marginal lungs. The establishment of EVLP in large transplant centers has favored an increase in the number of lung transplants, both by increasing the diagnostic accuracy of lung function and by constituting an effective platform for the reconditioning of lung grafts. In this context, faced with ethical and logistical issues, as well as in the study of immunological factors associated with lung transplantation, the development of rodent EVLP models has become important, given their reliability, the possibility of genetic manipulation, and lower costs. This paper describes a protocol for establishing a rat EVLP model and shows the inflammatory profile associated with the perfused lungs. This will help propagate knowledge about the rat EVLP model, promoting our understanding of the biological responses associated with that revolutionary technique.

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...

Biology

Evaluation of Lung Function in an Ex vivo Murine Model

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be evaluated through various lung maneuvers that involve different oscillatory waveforms. When applied to the lungs, these maneuvers measure multiple variables, such as pressure, volume, and flow, based on the response to the waveforms. These signals are then computed and analyzed to determine parameters such as hysterisivity, resistance, compliance, tissue damping, and tissue elastance, providing a detailed assessment of overall lung function. The analysis of respiratory mechanics is particularly important in evaluating donor lungs for lung transplantation. The present protocol is the first of its kind, offering a comprehensive and reproducible stepwise method for assessing respiratory mechanics using an ex vivo murine model. It includes details on the selected animal model, lung recovery, storage and preservation, and experimentation using a forced oscillation technique-based system. Additionally, it outlines data analysis, clinical significance, and the applications of the forced oscillation technique in studying an ex vivo model.

The present protocol describes a stepwise method for analyzing the respiratory mechanics of an ex vivo murine model using the forced oscillation technique (FOT).

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be ...

Ex vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue

Source: Harrington, N.E. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62187">Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung.</a> J. Vis. Exp. (2021).&#160;
This video describes the protocol for developing an ex vivo pig lung model of bacterial biofilm on infected bronchiolar tissue. Biofilms are bacterial aggregates embedded in a matrix adherent to a surface. This model is used to study the antibiotic susceptibility of bacteria at different stages of biofilm formation.

Source: Harrington, N.E. et al., Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the ...

JoVE Encyclopedia of Experiments

Large Animal Models

Porcine

Ex vivo Lung Perfusion: A Technique for Pulmonary Research in a Rabbit Model

Source: Pacheco-Baltazar, A., et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62734">Isolated Lung Perfusion System in the Rabbit Model.</a> J. Vis. Exp. (2021).
This video shows an ex vivo technique for isolated lung perfusion. It is a tool for pulmonary research that will aid studies of various physiological and pathological mechanisms involved in lung transplantation procedures.

Source: Pacheco-Baltazar, A., et al., Isolated Lung Perfusion System in the Rabbit Model. J. Vis. Exp. (2021).
This video shows an ex vivo technique for ...

Leporine

Ex Vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue

Place the bronchiole in the first DMEM/RPMI 1640 wash. Leave it in the wash, and harvest additional sections of bronchiole from the same lung to yield sufficient tissue sections for the planned experiment. Place any additional bronchiolar sections from the same lung into the wash, and leave them in the wash for at least two minutes.
Remove the bronchioles from the first wash and place the samples in a sterile Petri dish. Lightly, hold each bronchiole with sterile forceps, taking care not to damage the tissue. Remove as much remaining soft tissue as possible, and cut the tissue into five-millimeter wide strips using sterile dissection scissors.
Place all of the bronchiolar tissue strips into the second DMEM/RPMI 1640 wash. Leave them in the wash for at least two minutes. Then, remove the tissue strips from the second wash using sterile forceps, and place them in a clean, sterile Petri dish. Remove any remaining soft tissue attached to the bronchiole, and cut the strips into squares using sterile dissection scissors.
Add the third DMEM/RPMI 1640 wash into the Petri dish, and lightly mix the tissue pieces in the wash by swirling the dish. Pour the third wash out of the Petri dish without removing the tissue pieces.
Then, add the final SCFM wash, ensuring that all of the tissue pieces are covered. Sterilize the tissue in SCFM under UV light for five minutes. Use sterile forceps to transfer each sterilized bronchiolar tissue piece into individual wells of a 24-well plate containing SCFM agarose pads.
To infect each tissue piece with the desired bacterial strain, touch a colony grown on an agar plate with the tip of a 29-gauge needle attached to a sterile 0.5-milliliter insulin syringe. Then, touch the colony onto the tissue piece, gently pricking the surface. For the uninfected controls, gently prick the surface of the tissue piece with the tip of the needle.
Then, use a pipette to add 500 microliters of SCFM to each well. Sterilize a breathable sealing membrane for each 24-well plate under ultraviolet light for 10 minutes. Remove the lid from the 24-well plate, and replace it with the breathable membrane. Then, incubate the plates at 37 degrees Celsius without shaking.

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

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