The authors would like to thank Ph.D. Bettina Sommer Cervantes for her support in the writing of this manuscript, and Kitzia Elena Lara Safont for her support with the illustrations.

The isolated perfusion system in the rabbit model has been widely used in the Bronchial Hyperresponsiveness Laboratory at the Instituto Nacional de Enfermedades Respiratorias. The protocol includes New Zealand rabbits with an approximate weight of 2.5-3 kg. All animals were kept in standard vivarium conditions and ad libitum feeding in compliance with the official Mexican guidelines for laboratory animals (NOM 062-ZOO-1999) and under the Guide for the Care and Use of Laboratory Animals (8th edition, 2011). All the animal procedures and animal care methods presented in this protocol were previously approved by the Ethics Committee of the Instituto Nacional de Enfermedades Respiratorias.
NOTE: The preparation of the isolated lung perfusion system involves the deliberate death of an animal under anesthesia and via euthanasia.
1. Equipment and preparation of apparatus.
<ol>
	<li>Equipment arrangement:
	<ol>
		<li>Set up an operating table with size according to the weight of the rabbit.</li>
		<li>Mount the cover of the artificial thorax on the steel column with the glass chamber underneath and the ventilator with a roller pump by the sides.</li>
		<li>Ensure that the cover is easily inclined to have the tracheal cannula in line with the trachea to allow a faster connection.</li>
	</ol>
	</li>
	<li>Artificial thorax: 
	NOTE: It is an essential part of the system. It consists of a water-jacketed glass chamber sealed by a special cover. The cover works as the organ holder with the connections to cannulate the trachea and vessels embedded in it.
	<ol>
		<li>Set up a venturi jet operated by compressed air to generate the negative pressure inside the artificial thorax. 
		NOTE: The ventilation control module (VCM) allows separate adjustments of inspiratory and end-expiratory pressures as well as respiration rate and the ratio of inspiratory duration to total cycle duration.</li>
	</ol>
	</li>
	<li>Apparatus:
	<ol>
		<li>Ensure that a normally working apparatus consists of a main steel column mounted on a base plate holding the artificial thorax, with the pneumotachometer and weight transducer located above it and behind the preheating coil with a bubble trap.</li>
		<li>Connect one differential pressure transducer to the pneumotachometer and another to the chamber pressure. Set a different couple of pressure transducers behind the thorax to measure perfusion and venous pressures.</li>
		<li>Connect the changeover stock below the oxygenator with a level electrode and the ventilation system beside the apparatus.</li>
	</ol>
	</li>
</ol>
2. Surgical extraction of the cardiopulmonary block.
<ol>
	<li>Anesthesia:
	<ol>
		<li>Use a combination of a sedative (xylazine) and a barbiturate (pentobarbital). 
		NOTE: Different anesthetic cocktails can be used with no effect on experimental outcomes.</li>
		<li>First, sedate the healthy New Zealand rabbits with a single intramuscular injection of xylazine hydrochloride (3-5 mg/kg). Ensure that the rabbits remain calm and relaxed to allow further manipulation after a few minutes of the injection.</li>
		<li>Following sedation, use the marginal (lateral) ear veins as access to anesthetize the rabbits with an intravenous injection of pentobarbital sodium (28 mg/kg).</li>
	</ol>
	</li>
	<li>Monitoring:
	<ol>
		<li>To avoid insufficient anesthesia or excessive depression of cardiac and respiratory functions, monitor the following parameters. To assess the depth of anesthesia, perform a toe pinch test.</li>
		<li>Ensure that the mucous membrane is pink. Blue or gray shades indicate hypoxia.</li>
		<li>Ensure that the heart rate is between 120-135 beats/min, and that the body temperature does not drop below 36.5 &#176;C.</li>
	</ol>
	</li>
	<li>Animal placement:
	<ol>
		<li>Shave the rabbit&#39;s torso and place the animal on the operating table in supine position. Place the ventilation system near the table, behind the rabbit&#39;s head, to permit connecting the cannulae quickly after tracheotomy to avoid tissular damage.</li>
	</ol>
	</li>
	<li>Incision and tracheotomy:
	<ol>
		<li>Dissect the skin with a ventral median line incision of 3-5 cm from the diaphragm up to the neck.</li>
		<li>With the operating scissors, cut the anterior 2/3 of the trachea between two cartilage rings to insert the tracheal cannula through the tracheal fibrous membrane.</li>
		<li>Insert a 5 mm (outer diameter; OD) tracheal cannula through the tracheal fibrous membrane and use a 4-0 silk suture to fix it carefully.</li>
		<li>Place either forceps or tweezers underneath the trachea to ensure the cannula did not bend against the trachea.</li>
	</ol>
	</li>
	<li>Positive-pressure ventilation:
	<ol>
		<li>As long as the lungs remain outside the artificial thorax, use a small species respiration pump to ventilate a positive pressure in order to avoid lung collapse during the surgery.</li>
		<li>Initiate ventilation through the tracheal cannula connected to the respiration pump quickly after tracheotomy and before the thorax is opened.</li>
		<li>Set the tidal volume at 10 mL/kg. 
		NOTE: Depending on the experiment setup and artificial thorax model, provide positive-pressure ventilation by either the same ventilation pump used to provide negative-pressure or a different one, granting a quick re-cannulation.</li>
	</ol>
	</li>
	<li>Thoracotomy and exsanguination:
	<ol>
		<li>To access the thoracic cavity, use a scalpel or scissors to open the thorax wall and perform a medial sternotomy up to the upper third of the thorax.</li>
		<li>Hold the thorax halves open by two retractors. Several lung flaps usually surround the heart.</li>
		<li>Localize the superior and inferior vena cava and refer them with threads.</li>
		<li>Prior to the exsanguination of the animal, identify the right ventricle and inject 1000 UI/kg of heparin.</li>
		<li>Immediately after the injection, ligate the superior and inferior vena cava with the pre-looped thread and perform exsanguination.</li>
	</ol>
	</li>
	<li>Heart-lung harvest:
	<ol>
		<li>Harvest the cardiopulmonary block gently and quickly. Use direct digital dissection or spring scissors to separate the connective tissue so as to remove the lungs from the thorax.</li>
		<li>Dissect the vasculature in the area, as well as the esophagus.</li>
		<li>Cut through the manubrium sterni to extend the medial sternotomy towards the tracheal cannula, releasing the trachea on both sides from connecting tissue.</li>
		<li>Now, resect the trachea above the tracheal cannula. Gently pull up the cannula in a craniocaudal axis as the dorsal fixation of the trachea and lungs is resected.</li>
	</ol>
	</li>
	<li>Cannulation:
	<ol>
		<li>Lift the isolated lungs out of the thorax and carefully place them over a sterile gauze on a petri dish.</li>
		<li>To prevent atelectasis, ventilate the lungs using positive-pressure ventilation with positive end-expiratory pressure (PEEP) set at 2 cmH2O.</li>
		<li>Remove the ventricles by cutting them off the heart at the level of the atrioventricular groove.</li>
		<li>After opening the two ventricles, introduce the OD 4.6 mm pulmonary artery cannula for the rabbit with a basket through the pulmonary artery and introduce the OD 5.9 mm left atrium cannula for the rabbit with the basket through the mitral valve into the left atrium.</li>
		<li>Use a 4-0 silk suture in the pulmonary artery and left atrium to fix the cannulaes. Include the surrounding tissues in the ligatures of the pulmonary artery and left atrium to avoid the distension of these structures.</li>
		<li>Inject 250 mL of saline isotonic solution through the arterial cannulae to flush the remaining blood from the vascular bed.</li>
	</ol>
	</li>
</ol>
3. Perfusion technique.
<ol>
	<li>Setup:
	<ol>
		<li>Place the isolated lungs carefully into the lung chamber.</li>
		<li>Attach the trachea to the transductor on the cover of the chamber.</li>
		<li>Connect the cannulated vessels to the perfusion system.</li>
		<li>Close the chamber and secure it with the rotary lock. 
		NOTE: The recirculating perfusion circuit consists of an open venous reservoir, a peristaltic pump, a heat exchanger, and a bubble trap.</li>
		<li>At this point, attach the chamber lid and switch over a stopcock to switch from positive to negative pressure ventilation. To check the negative pressure ventilation of the lungs and airtight closure of the chamber, inspect the respiratory excursion of the lung and chamber pressure on the pressure gauge.</li>
		<li>Perfuse the lungs with 200 mL of artificial blood-free perfusate (a Krebs-Ringer bicarbonate buffer containing 2.5% of bovine albumin).</li>
		<li>Start the perfusate flow at 3 mL/min/kg, then slowly step up the flow over a 5-min period to 5 mL/min/kg. Reach a flow of 8 mL/min/kg over the next 5 min and then after another 5-min period reach a maximum flux of 10 mL/min/kg. Take care of avoiding air from getting into the circuit. 
		NOTE: Maintain the pH and the temperature of the perfusate within physiological ranges (pH 7.4-7.5; temperature, 37 &#176;C-38 &#176;C). To adjust the pH, add NaHCO3&#160;(1N) or increase the flow of carbon dioxide. Alternatively, use HCl (0.1N) to acidify.</li>
	</ol>
	</li>
	<li>Parameters:
	<ol>
		<li>Check whether the predetermined perfusion and ventilation parameters are set as required.</li>
		<li>Ventilate the lungs with humidified air at a frequency of 30 bpm, a tidal volume of 10 mL/kg, and an end-expiratory pressure (Pe) of 2 cmH2O. 
		NOTE: The pulmonary arterial pressure (0-20 mmHg) corresponds to the height of the liquid level in the oxygenator or reservoir in centimeters above the pulmonary trunk, while the pulmonary venous pressure corresponds to the height of the pressure equilibration chamber above the left atrium. Both values can be modified. Note that left atrium pressure is also 0-20 mmHg.</li>
	</ol>
	</li>
	<li>Achieving zone 3 conditions:
	<ol>
		<li>Use the two catheters connected to side ports of the cannulae secured in the pulmonary artery, left atrium, and pressure transducers to measure the arterial (Pa) and venous (Pv) pressures.</li>
		<li>Set the baseline pressures at the level of the lung hilum (Zero-reference).</li>
		<li>Conduct the experiments under zone 3 ventilation conditions. To achieve this, wait for 10-15 min to obtain an equilibrium characterized by an isogravimetric state.</li>
		<li>Ensure that the venous pressure is higher than the alveolar pressure (Palv) and the arterial pressure remains higher than both (Pa &#62; Pv &#62; Palv) for Zone 3 conditions to occur.</li>
		<li>Ensure that the lungs&#39; weight remains constant and arterial and left atrial pressures are stable to achieve zone 3 conditions to open up a maximum number of pulmonary vessels and maintain the microvascular bed content during the experiment. 
		NOTE: The measurement of Kfc as an indicator of pulmonary edema has no variation between a manual and an automatic perfusion system.</li>
	</ol>
	</li>
	<li>Electronic control and signal processing: Ensure that the respiratory flow, weight changes, microvascular pressure, tidal volume, vascular resistance, among others, are registered on a multiple central electronics unit that integrates signals coming from the transducers and displays them on the evaluation system.</li>
</ol>

<ol><li>Dixon, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+to+the+physiology+of+the+lungs%3A+Part+I.+The+bronchial+muscles%2C+their+innervation%2C+and+the+action+of+drugs+upon+them%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Contributions to the physiology of the lungs: Part I. The bronchial muscles, their innervation, and the action of drugs upon them.</a> The Journal of Physiology. 29 (2), 97-173 (1903).</li>
<li>Nelson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research%5BTitle%5D)+AND+%22World+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World Journal of Experimental Medicine. 4 (2), 7-15 (2014).</li>
<li>Roman, M. A., Nair, S., Tsui, S., Dunning, J., Parmar, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%3A+a+comprehensive+review+of+the+development+and+exploration+of+future+trends%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Ex vivo lung perfusion: a comprehensive review of the development and exploration of future trends.</a> Transplantation. 96 (6), 509-518 (2013).</li>
<li>Delaunois, A., Gustin, P., Ansay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+muscarinic+receptor+subtypes+mediating+pulmonary+oedema+in+the+rabbit%5BTitle%5D)+AND+%22Pulmonary+Pharmacology%22%5BJournal%5D)">Multiple muscarinic receptor subtypes mediating pulmonary oedema in the rabbit.</a> Pulmonary Pharmacology. 7 (3), 185-193 (1994).</li>
<li>Delaunois, A., Gustin, P., Vargas, M., Ansay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effect+of+various+antagonists+of+inflammatory+mediators+against+paraoxon-induced+pulmonary+edema+in+the+rabbit%5BTitle%5D)+AND+%22Toxicology+and+Applied+Pharmacology%22%5BJournal%5D)">Protective effect of various antagonists of inflammatory mediators against paraoxon-induced pulmonary edema in the rabbit.</a> Toxicology and Applied Pharmacology. 132 (2), 343-345 (1995).</li>
<li>Barr, H. A., Nicholas, T. E., Power, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+alveolar+surfactant+in+rats+at+rest+and+during+prolonged+hyperpnoea%3A+pharmacological+evidence+for+two+tissue+pools+of+surfactant%5BTitle%5D)+AND+%22British+Journal+of+Pharmacology%22%5BJournal%5D)">Control of alveolar surfactant in rats at rest and during prolonged hyperpnoea: pharmacological evidence for two tissue pools of surfactant.</a> British Journal of Pharmacology. 93 (3), 473-482 (1988).</li>
<li>Machuca, T. N., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Journal+of+Thoracic+Disease%22%5BJournal%5D)">Ex vivo lung perfusion.</a> Journal of Thoracic Disease. 6 (8), 1054-1062 (2014).</li>
<li>Steen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+human+transplantation+of+a+nonacceptable+donor+lung+after+reconditioning+ex+vivo%5BTitle%5D)+AND+%22The+Annals+of+Thoracic+Surgery%22%5BJournal%5D)">First human transplantation of a nonacceptable donor lung after reconditioning ex vivo.</a> The Annals of Thoracic Surgery. 83 (6), 2191-2194 (2007).</li>
<li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technique+for+prolonged+normothermic+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22The+Journal+of+Heart+and+Lung+Transplantation%3A+The+Official+Publication+of+the+International+Society+for+Heart+and+Lung+Transplantation%22%5BJournal%5D)">Technique for prolonged normothermic ex vivo lung perfusion.</a> The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart and Lung Transplantation. 27 (12), 1319-1325 (2008).</li>
<li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normothermic+ex+vivo+lung+perfusion+in+clinical+lung+transplantation%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> New England Journal of Medicine. 364 (15), 1431-1440 (2011).</li>
<li>Kao, C. C., Parulekar, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+perfusate+exchange+during%5BTitle%5D)+AND+%22Annals+of+Translational+Medicine%22%5BJournal%5D)">Is perfusate exchange during.</a> Annals of Translational Medicine. 8 (3), 43(2020).</li>
<li>Alquicira-Mireles, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Participaci%C3%B3n+de+la+serotonina+en+los+cambios+de+permeabilidad+vascular+en+la+preservaci%C3%B3n+pulmonar+en+conejo%22">Participación de la serotonina en los cambios de permeabilidad vascular en la preservación pulmonar en conejo</a>. , Universidad Nacional Autónoma de México. Biología thesis (2013).</li>
<li>Arreola-Ramírez, J. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Papel+de+la+liberaci%C3%B3n+de+acetilcolina+y+sustancia+P+en+el+deterioro+de+la+funci%C3%B3n+pulmonar+en+un+modelo+experimental+de+preservaci%C3%B3n+pulmonar+en+conejo%22">Papel de la liberación de acetilcolina y sustancia P en el deterioro de la función pulmonar en un modelo experimental de preservación pulmonar en conejo</a>. , Universidad Nacional Autónoma de México. Doctorado en Ciencias Biomédicas thesis (2009).</li>
<li>Isolated lung perfusion systems for small to large animal models. Harvard Apparatus. Hugo Sachs Elektronik (HSE). , Available from: 
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<li>Cowan, P. J., Hawthorne, W. J., Nottle, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenogeneic+transplantation+and+tolerance+in+the+era+of+CRISPR-Cas9%5BTitle%5D)+AND+%22Current+Opinion+in+Organ+Transplantation%22%5BJournal%5D)">Xenogeneic transplantation and tolerance in the era of CRISPR-Cas9.</a> Current Opinion in Organ Transplantation. 24 (1), 5-11 (2019).</li>
<li>Collaborators, G. C. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+and+attributable+health+burden+of+chronic+respiratory+diseases%2C+1990-2017%3A+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2017%5BTitle%5D)+AND+%22The+Lancet+Respiratory+Medicine%22%5BJournal%5D)">Prevalence and attributable health burden of chronic respiratory diseases, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.</a> The Lancet Respiratory Medicine. 8 (6), 585-596 (2020).</li>
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</ol>

The authors declare no conflicts of interest.

This work displays a general view of the isolated lung perfusion system, an essential technique in pulmonary physiology research. The isolated lung perfusion system offers a great degree of versatility in its uses and allows the evaluation of several parameters relevant in the testing of a wide range of hypotheses15. An isolated lung system is a tool with worldwide presence that, in the last decade, has further established its relevance for organ-specific evaluations and also expanded its usefulne...

Brodie and Dixon first described the ex-vivo lung perfusion system in 1903 1. Since then, it has become a gold standard tool for studying the physiology, pharmacology, toxicology, and biochemistry of the lungs2,3. The technique offers a consistent and reproducible way to evaluate the viability of lung transplants, and to determine the effect of inflammatory mediators such as histamine, arachidonic acid metabolites, and substance P, among others, as well as their interactions during pulmonary phenomena such as bronchoconstriction, atelectasis, and pulmonary edema. The isolated lung system has been a key technique in unveiling the important role of the lungs in the elimination of biogenic amines from general circulation4,5. Additionally, the system has been used to evaluate the biochemistry of pulmonary surfactant6. Over the last few decades, the ex-vivo lung perfusion system has become an ideal platform for lung transplantation research7. In 2001 a team lead by Stig Steen described the first clinical application of the ex-vivo lung perfusion system by using it to recondition the lungs of a 19-year-old donor, who was initially rejected by transplantation centers due to its injuries. The left lung was harvested and perfused for 65 min; afterward, it was successfully transplanted into a 70-year-old man with COPD8. Further research into lung reconditioning using the ex-vivo perfusion led to developing the Toronto technique for extended lung perfusion to assess and treat injured donor lungs9,10. Clinically, the ex-vivo lung perfusion system has shown to be a safe strategy to increase donor pools by treating and reconditioning sub-standard donor lungs, presenting no significant difference in risks or outcomes against standard criteria donors10. 
The main advantage of the isolated lung perfusion system is that the experimental parameters can be evaluated in a complete functional organ that preserves its physiological function under an artificial laboratory setup. Furthermore, it allows the measurement and manipulation of pulmonary mechanical ventilation to analyze the components of pulmonary physiology such as airway resistance, total vascular resistance, gas exchange, and edema formation, which to date cannot be measured precisely in vivo on lab animals2. Notably, the composition of the solution with which the lung is perfused can be fully controlled, enabling the addition of substances to evaluate their effects in real-time and sample collection from perfusion for further study11. Researchers working with the isolated lung system should bear in mind that mechanical ventilation causes decay of the pulmonary tissue shortening its useful time. This progressive fall in mechanical parameters can be significantly delayed by hyperinflating the lungs occasionally during the time of the experiment4. Still, the preparation cannot usually last more than eight hours. Another consideration for the ex-vivo lung perfusion system is the absence of central nervous regulation and lymphatic drainage. The effects of their absence are not yet fully understood and could potentially be a source of bias in certain experiments. 
The isolated lung perfusion system technique can be performed in the rabbit model with a high degree of consistency and reproducibility. This work describes the technical and surgical procedures for the implementation of the ex-vivo isolated lung perfusion technique as developed for the rabbit model at Instituto Nacional de Enfermedades Respiratorias in Mexico City, intending to share the insights and provide a clear guide on key steps in the application of this experimental model.

<table><tbody><tr><td>2-Stop Tygon E-Lab Tubing, 3.17 mm ID, 12/pack, Black/White</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-1864</td><td></td></tr><tr><td>Adapter for Positive Pressure Ventilation on IPL-4</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4312</td><td></td></tr><tr><td>Adapter for Positive Pressure Ventilation on IPL-4</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4312</td><td></td></tr><tr><td>Alternative Pressure-Free Gas Supply for IPL-4: To supply the trachea with gas mixture different from room air during negative ventilation</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4309</td><td></td></tr><tr><td>Base Unit for the Rabbit to Fetal Pig Isolated Perfused Lung</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4138</td><td></td></tr><tr><td>Bovine serum A2:D41albumin lyophilized powder</td><td>sigma</td><td>3912</td><td>500 g</td></tr><tr><td>Calcium chloride, CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O.</td><td>JT Baker</td><td>10035-04-8</td><td></td></tr><tr><td>Cryogenic vials</td><td>Corning</td><td>430659</td><td>2 mL</td></tr><tr><td>D-glucosa, C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;.</td><td>sigma</td><td>G5767</td><td></td></tr><tr><td>Differential Low Pressure Transducer DLP2.5, Range +- 2.5 cmH&lt;sub&gt;2&lt;/sub&gt;O, HSE Connector</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-3882</td><td></td></tr><tr><td>Differential Pressure Transducer MPX, Range +- 100 cmH&lt;sub&gt;2&lt;/sub&gt;O, HSE Connector</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0064</td><td></td></tr><tr><td>Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>Ethanol absolute HPLC grade</td><td>Caledon</td><td></td><td></td></tr><tr><td>Falcon tubes</td><td></td><td></td><td>14 mL</td></tr><tr><td>Harvard Peristaltic Pump P-230 (Complete with Control Box and P-230 Motor Drive)</td><td>Hugo Sachs Elektronik (HSE)</td><td>70-7001</td><td></td></tr><tr><td>Heated Linear Pneumotachometer 0 to 10 L/min flow range</td><td>Hugo Sachs Elektronik (HSE)</td><td>59-9349</td><td></td></tr><tr><td>Heater Controller for Single Pneumotachometer 230 VAC, 50 Hz</td><td>Hugo Sachs Elektronik (HSE)</td><td>59-9703</td><td></td></tr><tr><td>Heparin</td><td>PISA</td><td></td><td>5000 UI</td></tr><tr><td>HPLC Column (C18 100A 5U)</td><td>Alltech</td><td>98121213</td><td>150 mm x 4.6 mm</td></tr><tr><td>Hydrophilic Syringe Filter</td><td>Millex</td><td>SLLGR04NL</td><td>4 mm</td></tr><tr><td>IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4296</td><td></td></tr><tr><td>IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 V</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4296</td><td></td></tr><tr><td>Jacketed Glass Reservoir for Buffer Solution, with Frit and Tubing, 6.0 L</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0322</td><td></td></tr><tr><td>Lauda Thermostatic Circulator, Type E-103, 230 V/50 Hz, 3 L Bath Volume, Temperature Range 20 to 150&amp;deg;C</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0125</td><td></td></tr><tr><td>Left Atrium Cannula for Rabbit with Basket, OD 5.9 mm</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4162</td><td></td></tr><tr><td>Low Range Blood Pressure Transducer P75 for PLUGSYS Module</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0020</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>JT Baker</td><td>10034-99-8</td><td></td></tr><tr><td>Microcentrifuge Tube</td><td>Corning</td><td>430909</td><td></td></tr><tr><td>Negative Pressure Ventilation Control Option with Pressure Regulator for IPL-4</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4298</td><td></td></tr><tr><td>New Zeland rabbits</td><td></td><td></td><td></td></tr><tr><td>PISABENTAL (Pentobarbital sodium)</td><td>PISA</td><td>Q-7833-215</td><td></td></tr><tr><td>PLUGSYS Case, Type 603* 7</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0045</td><td></td></tr><tr><td>PLUGSYS TCM Time Counter Module</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-1750</td><td></td></tr><tr><td>PLUGSYS Transducer Amplifier Module (TAM-A)</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-0065</td><td></td></tr><tr><td>PLUGSYS Transducer Amplifier Module (TAM-D)</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-1793</td><td></td></tr><tr><td>PLUGSYS VCM-4R Ventilation Control Module with Pressure Regulator</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-1755</td><td></td></tr><tr><td>Potassium chloride, KCl.</td><td>JT Baker</td><td>3040-01</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>JT Baker</td><td>7778-77-0</td><td></td></tr><tr><td>PROCIN (Xylacine clorhydrate)</td><td>PISA</td><td>Q-7833-099</td><td></td></tr><tr><td>Pulmonary Artery Cannula for Rabbit with Basket, OD 4.6 mm</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4161</td><td></td></tr><tr><td>Scalpel knife</td><td></td><td></td><td></td></tr><tr><td>Serotonin 5-HT</td><td></td><td></td><td></td></tr><tr><td>Servo Controller for Perfusion (SCP</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-2806</td><td></td></tr><tr><td>Snap Cap Microcentrifuge Tube</td><td>Costar</td><td>3620</td><td>1.7 mL</td></tr><tr><td>Sodium bicarbonate, NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>sigma</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride, NaCl.</td><td>sigma</td><td>S9888</td><td></td></tr><tr><td>Surgical gloves No. 7 1/2</td><td></td><td></td><td></td></tr><tr><td>Surgical gloves No. 8</td><td></td><td></td><td></td></tr><tr><td>Taygon tubes</td><td>Masterflex</td><td></td><td></td></tr><tr><td>Tracheal Cannula for Rabbit, OD 5.0 mm</td><td>Hugo Sachs Elektronik (HSE)</td><td>73-4163</td><td></td></tr></tbody></table>

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 lung perfusion system allows organ manipulation for biopsy, sample collection from perfusion, and real-time data collection of physiological parameters. The isolated system can be used to test many hypotheses involving different functions and lung phenomena, from metabolic and enzymatic activity to edema formation and preservation periods for lung transplants.
Figure 1&#160;displays a diagram of the fully assembled isolated lung perfusion system along...

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Isolated Lung Perfusion System in the Rabbit Model

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 ...

isolated-lung-perfusion-system-in-the-rabbit-model

Research

JoVE Journal

Medicine

3.6K Views.  Instituto Nacional de Enfermedades Respiratorias. Deze techniek is nuttig bij de karakterisering van longfysiologie en pathologie door metabole activiteiten en ademhalingsfunctie te meten. Geïsoleerd longperfusiesysteem maakt het mogelijk om te werken met een compleet functioneel orgaan dat de studie van een continue fysiologische functie mogelijk maakt terwijl ventilatie en perfusie worden gerecreëerd. Deze methode zou inzicht kunnen geven in de karakterisering van de niet-respiratoire mogelijkheden van de longweefsels, zoals metabole act...

Video: Isolated Lung Perfusion System in the Rabbit Model

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Biology

Development and Validation of a Porcine Ex Situ Isolated Liver Perfusion Model 

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

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 ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

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 ...

Cardiopulmonary Block Harvest from Rabbit: A Surgical Procedure to Retrieve Functional Heart-Lung Block from Rabbit Model

Source: Pacheco-Baltazar, A., et al. <a href="https://www.jove.com/v/62734">Isolated Lung Perfusion System in the Rabbit Model.</a> J. Vis. Exp. (2021)
In this video, we show a procedure to surgically harvest a functional cardiopulmonary block from a rabbit model. The harvested block can be used for further physiological, pharmacological or biochemical studies.

Source: Pacheco-Baltazar, A., et al. Isolated Lung Perfusion System in the Rabbit Model. J. Vis. Exp. (2021)
In this video, we show a procedure to ...

JoVE Encyclopedia of Experiments

Large Animal Models

Leporine

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/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 ...

Isolated Lung Perfusion System in the Rabbit Model

Samenvatting

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