We thank FAPESP (Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo) for granting financial support.

Animal experiments complied with the Ethics Committee for Experimental Animals Use and Care of the Faculty of Medicine of the University of S&#227;o Paulo (protocol number 112/16).
1. Animal grouping
<ol>
	<li>Randomly assign twelve male Sprague Dawley rats (250-300 g) to one of three experimental groups (n=4) to analyze and compare the effects associated with the animal models.</li>
	<li>Assign animals to hemorrhagic shock group (HS, n=4): animals subjected to vascular catheterization with hemorrhagic shock induction + maintenance for 360 min + cardiopulmonary block extraction + sample preparation for analysis.</li>
	<li>Assign animals to brain death group (BD, n=4): animals subjected to brain death + maintenance for 360 min + cardiopulmonary block extraction + sample preparation for analysis.</li>
	<li>Assign animals to circulatory death group (CD, n=4): animals subjected to vascular catheterization + induction of circulatory death + suspension of ventilation + ischemia at room temperature for 180 min + sample preparation for analysis.</li>
</ol>
2. Anesthesia and presurgical preparation
<ol>
	<li>Place the rat in a closed chamber with 5% isoflurane for 1&#160;- 4 min. Confirm proper anesthetization by checking the toe pinch reflex. In the absence of reflex reactions (no paw retraction), perform orotracheal intubation (14-G angiocath) with the aid of a pediatric laryngoscope.</li>
	<li>With a previously adjusted mechanical ventilator (FiO2 100%, tidal volume 10 mL/kg, 90 cycles/min, and PEEP 3.0 cmH2O), connect the tracheal catheter to the ventilator, and adjust the anesthetic concentration to 2%. 
	NOTE: All procedures related to animal models followed the same anesthetic protocol described in this section.</li>
	<li>Remove fur from the regions of interest (head, neck, chest and abdomen). Then, using gauze, disinfect the surgical field and the animal&#39;s tail. Disinfection is performed with three alternating rounds of an alcoholic solution of chlorhexidine digluconate scrub.</li>
	<li>Cut the tip of the animal&#39;s tail, place the thumb and the index finger over the base of the tail, and then press and slide them away from the base. Collect a peripheral blood sample (20 &#181;L) through the tail for the total leukocyte count8. 
	NOTE: This procedure must be performed before the start of the tracheostomy and immediately at the end of each protocol (BD and HS - after 360 min).</li>
	<li>Use a precision pipette to dilute the collected blood in 380 &#181;L (1:20) of Turk&#39;s solution (Glacial acetic acid 99%). Once diluted, pipette the blood sample into a Neubauer chamber and place it under a microscope (40x). Perform the total leukocyte count in the four lateral quadrants of the chamber.</li>
</ol>
3. Tracheostomy
<ol>
	<li>With the help of appropriate scissors and forceps, perform longitudinal dissection of the cervical trachea, starting from the middle third of the neck to the suprasternal notch (<img alt="Equation 1" src="/files/ftp_upload/62975/62975eq01.jpg" style="margin: auto;" />1.5 cm incision). After the incision of the skin and subcutaneous tissue, dissect the cervical muscles until the trachea is exposed.</li>
	<li>Place one 2-0 silk ligature beneath the trachea.</li>
	<li>Using microscissors, tracheostomize the upper third of the trachea to achieve uniform ventilation. Horizontally cut the trachea between two cartilaginous rings to accommodate the diameter of a metal cannula (3.5 cm).</li>
	<li>Insert the ventilation tube and fix it with prepared ligatures.</li>
	<li>Connect the ventilation tube to the small-animal ventilation system.</li>
	<li>Ventilate the rat with a tidal volume of 10 mL/kg, rate of 70 cycles/min, and PEEP of 3 cmH2O.</li>
</ol>
4. Femoral artery and vein catheterization
<ol>
	<li>Expose the femoral triangle through a small incision (<img alt="Equation 1" src="/files/ftp_upload/62975/62975eq01.jpg" style="margin: auto;" />1.5 cm) in the inguinal region. Identify and isolate the femoral vessels. For this procedure, use a stereomicroscope (3.2x magnification).</li>
	<li>Place two 4-0 silk ligatures beneath the blood vessels (vein or artery), one distally and the other proximally. Close the most distal ligature, then place a preadjusted knot in the proximal ligature and pull.</li>
	<li>Insert the catheter through a small, pre-formed incision in the vessels. Fixate the cannula&#160;to avoid dislocation. 
	NOTE: Make the catheters from a 20 cm neonatal extender welded by heating to a peripheral intravenous catheter suitable for the caliber of the animal&#39;s venous network, thus preventing regurgitation of blood contents. Lubricate the cannula&#160;with heparin, avoiding the formation of thrombi and complications during mean arterial pressure (MAP) measurement.</li>
	<li>Connect the artery catheter to a pressure transducer and a vital sign monitoring system to record the mean arterial pressure (MAP). The transducer should be positioned at the level of the animal&#39;s heart. Record the MAP every 10-min period.</li>
	<li>Place the syringe catheter (3 mL) into the vein, aiming for hydration and exsanguination when necessary.</li>
</ol>
5. Hemorrhagic shock induction
<ol>
	<li>Through venous access and with a heparinized syringe, remove small volumes of blood until MAP values of <img alt="Equation 1" src="/files/ftp_upload/62975/62975eq01.jpg" style="margin: auto;" />50 mmHg are reached, thus establishing hemorrhagic shock. 
	NOTE: Collect a 2 mL aliquot of blood every 10 min in the first hour of the experiment and every 30 min in the subsequent hours.</li>
	<li>Keep the pressure stable at approximately 50 mmHg for a period of 360 min. To do so, remove or add aliquots of blood if the pressure increases or decreases, respectively.</li>
	<li>Put a source of heat nearby to avoid hypothermia. 
	NOTE: Here, a heat lamp is used.</li>
	<li>At the end of the protocol, harvest the pulmonary block at the total lung capacity (TLC) and either flash freeze in liquid nitrogen or place it in a fixing solution for further studies. 
	NOTE: With the aid of a small animal ventilator, the ventilatory parameters can be accessed during the protocol. In the present study, these parameters were evaluated immediately before HS induction (Baseline) and 360 min later (Final).</li>
</ol>
6. Circulatory death induction
<ol>
	<li>To induce circulatory death, administer 150 mg/kg sodium thiopental through the venous line. Then turn off the ventilation system.</li>
	<li>Note the progressive decrease in MAP until it reaches 0 mmHg. From this point, consider the start of the warm ischemia period and begin the time count. The animal should remain at room temperature (approximately 22 &#176;C) for 180 min.</li>
	<li>At the end of the protocol, reconnect the lungs to the mechanical ventilator and harvest the pulmonary block at TLC for collection. Either flash freeze using liquid nitrogen or place it in the fixation solution for further studies.</li>
</ol>
7. Brain death induction
<ol>
	<li>Place the rat in the prone position.</li>
	<li>Remove the skin from the skull using surgical scissors. Drill a 1 mm caliber borehole 2.80 mm anterior and 10.0 mm ventral to bregma and 1.5 mm lateral to the sagittal suture.</li>
	<li>Insert the entire balloon catheter into the cranial cavity and ensure that the balloon is prefilled with saline (500 &#181;L).</li>
	<li>With the help of a syringe, rapidly inflate the catheter.</li>
	<li>Confirm brain death by observing an abrupt MAP elevation (Cushing&#39;s reflex), the absence of reflexes, bilateral mydriasis, and apnea. After confirmation, discontinue anesthesia and keep the animal on mechanical ventilation for 360 min.</li>
	<li>Place a source of heat nearby to avoid hypothermia.</li>
	<li>At the end of the protocol, harvest the pulmonary block at TLC for collection and either flash freeze in liquid nitrogen or place it in a fixing solution for further studies. 
	NOTE: With the aid of a small animal ventilator, the ventilatory parameters can be accessed during the protocol. In the present study, we evaluated these parameters immediately before BD induction (Baseline) and after 360 minutes (Final).</li>
</ol>

We wish to confirm that there are no known conflicts of interest associated with this publication and that there has been no significant financial support for this work that could have influenced its outcome.

In recent years, the increasing number of diagnoses of brain death has led to it becoming the largest provider of organs and tissues intended for transplantation. This growth, however, has been accompanied by an incredible increase in donations after circulatory death. Despite its multifactorial nature, most of the triggering mechanisms of the causes of death begin after or accompany trauma with extensive loss of blood content4,18.
In this context, experimental models of brain death, circulatory arrest, and hemorrhagic shock are important tools for the prospective study of complications associated with the cause of donor death and their impact on the viability of potential organs intended for transplantation6,8,10. Several animal lineages have been suggested for model establishment, such as swine, rabbit, rat and mouse. Rat and mouse models are more common in the literature because they are not very expensive and involve low logistical difficulty while satisfactorily reproducing the pathophysiological events under study8,13,14,15.
We would like to emphasize that recent guidelines and studies have endorsed the use of pre-anesthetic analgesia as an integral part of surgical protocols, even in acute situations, aiming for more comprehensive management of perioperative pain and animal well-being. We recommend that researchers evaluate such an approach in future studies.
Brain death (BD) 
The BD model was found to be reproducible by means of an abrupt increase in ICP. The use of appropriate instruments and trained personnel allows surgical success and reproduction of the technique with a few weeks of training. During the development of the BD technique, trepanation should be performed with an appropriate motorized drill so that there is no slack in the catheter, thus preventing the projection of brain tissue out of the hole. In addition, during drilling, the forward movement of the drill should be stopped as soon as the initial resistance offered by the skull is overcome.
Researchers should remain alert and ensure rapid inflation of the catheter, as gradual inflation promotes distinct inflammatory and hemodynamic responses21. Blood pressure changes, in turn, should be monitored constantly throughout the protocol, especially during catheter insufflation, which should be accompanied by an abrupt increase in MAP and during the first hour after BD establishment (post-inflation hypotension period). These results are in agreement with the literature, which shows the establishment of a hypertensive peak immediately after catheter insufflation, followed by a decrease in pressure levels, in a likely response to the transient increase in circulating catecholamine levels22.
Maintaining the animal in BD for prolonged periods may lead to hypotension followed by circulatory death, making the experiment unfeasible. Accordingly, most protocols used in the literature establish a follow-up period that varies from 4 to 6 hours, after which vasoactive drugs must be administered12,13,21,22,23.
In addition to hemodynamic changes, cerebral infarction and ischemia promote an increase in the systemic circulation of proinflammatory factors, which, when they reach the lungs, contribute to lung parenchyma injury24,25,26.
In our study, BD was accompanied by a significant increase in tissue IL-1&#946; expression (over CD) and the wet/dry weight ratio, an index of pulmonary edema. Previous studies have indicated an increase in circulating levels of proinflammatory cytokines after a BD event, which may ultimately favor the modulation of the expression of adhesion molecules, increased vascular permeability, and consequent leukocyte migration27,28,29,30.
Hemorrhagic shock (HS) 
Established through the withdrawal or reinfusion of blood aliquots with the goal of prolonged hypotension maintenance (&#8804; 50 mmHg), the fixed pressure model of HS aims to mimic the decrease in blood volume caused by the hemorrhagic process and, consequently, the attenuation of the systemic filling pressure. These events lead to a decrease in MAP, accompanied by a decrease in pulmonary perfusion pressure31,32.
Among the advantages of this HS model is the possibility of controlling the degree and duration of hypotension, in addition to the greater reproducibility of the technique when compared to models based on a prefixed blood volume. Accordingly, most protocols used in the literature establish a protocol period that varies from 15 min to more than 180 min, with mean blood pressure levels ranging from 20-55 mmHg, depending on the analysis chosen in the study6,32. In the present study, hypotension was maintained for 3 hours, leading to increased tissue resistance, followed by decreased lung compliance in animals subjected to HS. Corroborating this, different studies in the literature have indicated a proportional relationship between the time spent in HS and the impacts of hypovolemia on airway resistance and lung compliance6,33,34.
In addition, in the present study, HS was accompanied by significant leukocytosis and increased tissue expression of IL-1&#946; (with respect to CD) and TNF-&#945;. Injury to the pulmonary microvasculature endothelium, induced by the release of reactive oxygen species from the primary process of hypoxia and established ischemia, will increase vascular permeability, which, together with the increase in pulmonary artery pressure, will act as a chemotactic factor for leukocytes and the subsequent release of inflammatory mediators6,20,31,35,36,37,38.
Circulatory death (CD) 
The main difference between the marginal grafts originating from the BD and CD processes is the warm ischemia time (WIT) to which the graft will be subjected, defined by some researchers as the time between the absence of peripheral pulses and interruption of blood flow due to removal of life support equipment until cold or regional perfusion of the organ17,39,40.
In the present study, the organs and tissues of animals derived from the CD model were subjected to a WIT period of 180 min. Several studies in the literature have revealed a proportional relationship between the WIT and post-transplantation dysfunction, suggesting that the ischemia time should vary according to the particularities and integrity of each organ. In this context, lung grafts from rats have been shown to tolerate up to 3-h periods of warm ischemia41,42.
With evidence of tissue injury caused by the predominant sympathetic phase, hemodynamic instability, and systemic inflammation resulting from the BD process, donations after circulatory arrest have been reconsidered as a potential strategy to decrease complications associated with transplantation41,42,43. In this sense, our data indicate a dramatic decrease in IL-1&#946; and TNF-&#945; levels in the CD model with respect to the other two models studied. Corroborating this, Iskender et al.4 noted the low levels of tissue cytokines in a model of lung reperfusion in rats with tissues donated after the WIT through mechanisms that are still poorly understood.
Based on the above, the choice of methodology and its adaptations should depend on the objectives developed by the researcher. Once determined, these objectives should guide the type of donation model, the protocol time and the analyses to be performed. It is also possible to relate the type of donation with animal models of lung reconditioning and reperfusion.
Conclusions 
In conclusion, the organ donor models described here are potential tools in the study of the changes associated with different graft harvesting methodologies and could provide means by which a full understanding of the impact of the quality of these organs on post-transplantation outcomes can be obtained, given the reproducibility and reliability of the methodologies presented here.

Clinical relevance 
Organ transplantation is a well-established therapeutic option for several serious pathologies. In recent years, many advances have been achieved in the clinical and experimental fields of organ transplantation, such as greater knowledge of the pathophysiology of primary graft dysfunction (PGD) and advances in the areas of intensive care, immunology, and pharmacology1,2,3. Despite the achievements and improvements in the quality of the related surgical and pharmacological procedures, the relationship between the number of available organs and the number of recipients on the waiting list remains one of the main challenges2,4. In this regard, the scientific literature has proposed animal models for studying therapies that can be applied to organ donors to treat and/or preserve the organs until the time of transplantation5,6,7,8.
By mimicking the different events observed in clinical practice, animal models allow the study of the associated pathological mechanisms and their respective therapeutic approaches. The experimental induction of these events, in most isolated cases, has generated experimental models of organ and tissue donation that are widely investigated in the scientific literature on organ transplantation6,7,8,9. These studies employ different methodological strategies, such as those inducing brain death (BD), hemorrhagic shock (HS), and circulatory death (CD), since these events are associated with different deleterious processes that compromise the functionality of the donated organs and tissues.
Brain death (BD) 
BD is associated with a series of events that lead to the progressive deterioration of different systems. It usually occurs when an acute or gradual increase in intracranial pressure (ICP) happens due to brain trauma or hemorrhage. This increase in ICP promotes an increase in blood pressure in an attempt to maintain a stable cerebral blood flow in a process known as Cushing&#39;s reflex10,11. These acute changes can result in cardiovascular, endocrine, and neurological dysfunctions that compromise the quantity and quality of the donated organs, in addition to impacting post-transplantation morbidity and mortality10,11,12,13.
Hemorrhagic shock (HS) 
HS, in turn, is often associated with organ donors, as most of them are victims of trauma with significant loss of blood volume. Some organs, such as the lungs and heart, are particularly vulnerable to HS due to hypovolemia and consequent tissue hypoperfusion14. HS induces lung injury through increased capillary permeability, edema, and infiltration of inflammatory cells, mechanisms that together compromise gas exchange and lead to progressive organ deterioration, consequently derailing the donation process6,14.
Circulatory death (CD) 
The use of post-CD donation has been growing exponentially in major world centers, thus contributing to the increase in the number of collected organs. Organs recovered from post-CD donors are vulnerable to the effects of warm ischemia, which occurs after an interval of low (agonic phase) or no blood supply (asystolic phase)8,15. Hypoperfusion or the absence of blood flow will lead to tissue hypoxia associated with the abrupt loss of ATP and the accumulation of metabolic toxins in tissues15. Despite its current use for transplantation in clinical practice, many doubts remain about the impact of the use of these organs on the quality of the post-transplant graft and on patient survival15. Thus, the use of experimental models for a better understanding of the etiological factors associated with CD is also growing8,15,16,17.
Experimental models 
There are various experimental organ donation models (BD, HS, and CD). However, studies often focus on only one strategy at a time. There is a noticeable gap in studies that combine or compare two or more strategies. These models are very useful in the development of therapies that seek to increase the number of donations and consequently decrease the waiting list of potential recipients. The animal species used for this purpose vary from study to study, with porcine models being more commonly selected when the objective is a more direct translation with human morpho physiology and less technical difficulty in the surgical procedure due to the size of the animal. Despite the benefits, logistical difficulties and high costs are associated with the porcine model. On the other hand, the low cost and possibility of biological manipulation favor the use of rodent models, allowing the researcher to start from a reliable model to reproduce and treat lesions, as well as to integrate the knowledge acquired in the field of organ transplantation.
Here, we present a rodent model of brain death, circulatory death, and hemorrhagic shock donation. We describe inflammatory processes and pathological conditions associated with each of these models.

<table><tbody><tr><td>14-gauge angiocath</td><td>DB</td><td>38186714</td><td>Orotracheal intubation</td></tr><tr><td>2.0-silk</td><td>Brasuture</td><td>AA553</td><td>Tracheal tube fixation</td></tr><tr><td>24-gauge angiocath</td><td>DB</td><td>38181214</td><td>Arterial and venous access</td></tr><tr><td>4.0-silk</td><td>Brasuture</td><td>AA551</td><td>Fixation of arterial and venous cannulas</td></tr><tr><td>Alcoholic chlorhexidine digluconate solution (2%).</td><td>Vic Pharma</td><td>Y/N</td><td>Asepsis</td></tr><tr><td>Trichotomy apparatus</td><td>Oster</td><td>&lt;strong&gt;Y/N&lt;/strong&gt;</td><td>&lt;strong&gt;Clipping device&lt;/strong&gt;</td></tr><tr><td>Precision balance</td><td>&lt;strong&gt;Shimadzu&lt;/strong&gt;</td><td>D314800051</td><td>Analysis of the wet/dry weight ratio</td></tr><tr><td>Barbiturate (Thiopental)</td><td>Crist&amp;aacute;lia</td><td>18080003</td><td>DC induction</td></tr><tr><td>Balloon catheter (Fogarty-4F)</td><td>Edwards Life Since</td><td>120804</td><td>BD induction</td></tr><tr><td>Neonatal extender</td><td>Embramed</td><td>497267</td><td>Used as catheters with the aid of the 24 G angiocath</td></tr><tr><td>FlexiVent</td><td>Scireq</td><td>1142254</td><td>Analysis of ventilatory parameters</td></tr><tr><td>Heparin</td><td>&lt;strong&gt;Blau Farmaceutica SA&lt;/strong&gt;</td><td>&lt;strong&gt;7000982-06&lt;/strong&gt;</td><td>&lt;strong&gt;Anticoagulant&lt;/strong&gt;</td></tr><tr><td>Isoflurane</td><td>Crist&amp;aacute;lia</td><td>10,29,80,130</td><td>Inhalation anesthesia</td></tr><tr><td>Micropipette (1000 &amp;micro;L)</td><td>&lt;strong&gt;Eppendorf&lt;/strong&gt;</td><td>&lt;strong&gt;347765Z&lt;/strong&gt;</td><td>&lt;strong&gt;Handling of small- volume liquids&lt;/strong&gt;</td></tr><tr><td>Micropipette (20 &amp;micro;L)</td><td>&lt;strong&gt;Eppendorf&lt;/strong&gt;</td><td>&lt;strong&gt;H19385F&lt;/strong&gt;</td><td>&lt;strong&gt;Handling of small- volume liquids&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>1601004545</td><td>Assistance in the visualization of structures for the surgical procedure</td></tr><tr><td>Multiparameter monitor</td><td>Dixtal</td><td>101503775</td><td>MAP registration</td></tr><tr><td>Motorized drill</td><td>Midetronic</td><td>MCA0439</td><td>Used to drill a 1 mm caliber borehole</td></tr><tr><td>Neubauer chamber</td><td>Kasvi</td><td>D15-BL</td><td>Cell count</td></tr><tr><td>Pediatric laryngoscope</td><td>Oxygel</td><td>Y/N</td><td>Assistance during tracheal intubation</td></tr><tr><td>Syringe (3 mL)</td><td>SR</td><td>3330N4</td><td>Hydration and exsanguination during HS protocol</td></tr><tr><td>Pressure transducer</td><td>Edwards Life Since</td><td>P23XL</td><td>MAP registration</td></tr><tr><td>Metallic tracheal tube</td><td>Biomedical</td><td>006316/12</td><td>Rigid cannula for analysis with the FlexiVent ventilator</td></tr><tr><td>Isoflurane vaporizer</td><td>Harvard Bioscience</td><td>1,02,698</td><td>Anesthesia system</td></tr><tr><td>Mechanical ventilator for small animals (683)</td><td>Harvard Apparatus</td><td>MA1 55-0000</td><td>Mechanical ventilation</td></tr><tr><td>xMap methodology</td><td>Millipore</td><td>RECYTMAG-65K-04</td><td>Analysis of inflammatory markers</td></tr></tbody></table>

study of experimental organ donation models for lung transplantation

Experimental models are important tools for understanding the etiological phenomena involved in various pathophysiological events. In this context, different animal models are used to study the elements triggering the pathophysiology of primary graft dysfunction after transplantation to evaluate potential treatments. Currently, we can divide experimental donation models into two large groups: donation after brain death and donation after circulatory arrest. In addition, the deleterious effects associated with hemorrhagic shock should be considered when considering animal models of organ donation. Here, we describe the establishment of three different lung donation models (post-brain death donation, post-circulatory death donation, and post-hemorrhagic shock donation) and compare the inflammatory processes and pathological disorders associated with these events. The objective is to provide the scientific community with reliable animal models of lung donation for studying the associated pathological mechanisms and searching for new therapeutic targets to optimize the number of viable grafts for transplantation.

Mean arterial pressure (MAP) 
To determine the hemodynamic repercussions of BD and HS, MAP was evaluated across the 360 min of the protocol. The baseline measurement was collected after skin removal and skull drilling and before blood aliquot collection for animals subjected to BD or HS, respectively. Prior to BD and HS induction, the baseline MAP of the two groups was similar (BD: 110.5 &#177; 6.1 vs. HS: 105.8 &#177; 2.3 mmHg; p=0.5; two-way ANOVA). After catheter insufflation, the BD group experienced an abrupt increase in blood pressure levels (138. 7 &#177; 10.1 mmHg). The hypertensive peak is a peculiar event related to increased intracranial pressure and can be considered the first evidence of the establishment of BD. In addition, we observed the absence of reflexes, bilateral mydriasis, and post-inflation apnea in all animals. This peak pressure was followed by a rapid decrease in MAP (10 min - 81.2 &#177; 10 mmHg). Hypotension persisted for approximately 50 min, after which MAP levels returned to values close to those at baseline (120 min - 120.7 &#177; 7.5 mmHg) (Figure 1).
Unlike in the BD group, the decrease in MAP in the HS group is associated with the withdrawal of blood aliquots in the first 10 min of the experiment. Hypovolemic shock was maintained for 360 min (mean variation throughout the protocol 52.3 &#177; 1.2 mmHg). After the end of the protocol, the BD group showed a significantly different MAP pattern over the 6-h follow-up from the HS group (BD: 93.7 &#177; 4.5 vs. HS: 52.3 &#177; 0.5 mmHg; p&#60;0.0001; Student&#39;s t test).
Pulmonary mechanics 
To evaluate the elastic and resistive parameters of the respiratory system, an analysis of the lung mechanics of the animals subjected to BD and HS was performed. 360 min after onset and after hypotension maintenance, the HS group exhibited increased lung tissue resistance (G) (HS: Baseline - 0.26 &#177; 0.02 vs. Final - 0.51 &#177; 0.05 cmH2O.mL-1; p=0.03; two-way ANOVA), followed by reduced respiratory system compliance (Crs) (HS: Baseline - 0.64 &#177; 0.05 vs. Final - 0.23 &#177; 0.004 cmH2O/mL; p=0.001; two-way ANOVA) (Figure 2A,B).
Pulmonary edema 
At the end of the protocol, the middle lobe of the right lung was collected for all groups, and its weight was measured to analyze the wet/dry weight ratio, which was used as the pulmonary edema index. The wet weight was assessed immediately after extraction of the organ, and the dry weight was measured after 24 h in an 80 &#176;C oven. According to this ratio, the BD group (2.32 &#177; 0.1) showed greater edema than the HS (1.97 &#177; 0.03) and CD groups (2.04 &#177; 0.02) (Figure 3).
Systemic and tissue inflammatory parameters 
At the end of the protocol, there was a significant increase in the total number of systemic leukocytes in the group that underwent HS (Baseline - 13888 &#177; 887.3 vs. Final - 35263 &#177; 4076 mm3; p=0.0189); two-way ANOVA) (Figure 4). The HS group also showed an increase in the number of leukocytes both when compared to the baseline values and in relation to the BD group (p = 0.0132).
Tissue inflammation was assessed by quantifying inflammatory markers in the lung tissue. For this purpose, lung tissue biopsy samples were homogenized in phosphate buffer and then sent for analysis for tumor necrosis factor alpha (TNF-&#945;) and interleukin 1 beta (IL1-&#946;) expression. IL1-&#946; expression levels were greater in the BD group (304.4 &#177; 91 pg/mg) and HS group (327.5 &#177; 25.2 pg/mg) than in the CD group (8 &#177; 2.3 pg/mg; p=0.004; one-way ANOVA) (Figure 5B). The HS group also showed higher levels of TNF-&#945; (4.7 &#177; 0.3 pg/mg; p&#60;0.0001; one-way ANOVA) than the BD group (1.3 &#177; 0.3 pg/mg) and CD group (0.4 &#177; 0.2 pg/mg) (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62975/62975fig01.jpg" /> 
Figure 1: Time course of mean arterial pressure (MAP) in the brain death (BD) and hemorrhagic shock (HS) groups. The values for all of the measurements are expressed as the means &#177; standard errors of the means (SEMs). MAP, mean arterial pressure; BD, brain death; HS, hemorrhagic shock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62975/62975fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62975/62975fig02.jpg" /> 
Figure 2: Lung mechanics. Lung mechanics as determined by (A) respiratory system compliance and (B) tissue resistance in the brain death (BD) group and the hemorrhagic shock (HS) group. * indicates significant differences between the baseline and final values in the HS group (p&#60;0.05). The values for all the measurements are expressed as the means &#177; standard errors of the means (SEMs), and two-way ANOVA was used for comparisons. Crs, compliance of the respiratory system; G, tissue resistance; BD, brain death; HS, hemorrhagic shock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62975/62975fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62975/62975fig03.jpg" /> 
Figure 3: Lung edema determined by lung wet-to-dry weight ratio in the brain death (BD) group and the hemorrhagic shock (HS) group. The values for all the measurements are expressed as the means &#177; standard errors of the means (SEMs), and comparisons were made with one-way ANOVA. BD, brain death; HS, hemorrhagic shock; CD, circulatory death. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62975/62975fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62975/62975fig04.jpg" /> 
Figure 4: Leukogram of the hemorrhagic shock (HS) group and brain death (BD) group. * indicates significant differences between baseline and final values in the HS group (p&#60;0.05). The values for all the measurements are expressed as the means &#177; standard errors of the mean (SEMs), and comparisons were made with two-way ANOVA. BD, brain death; HS, hemorrhagic shock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62975/62975fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62975/62975fig05.jpg" /> 
Figure 5: Local inflammatory responses were less prominent in the circulatory death (CD) group. (A) Lung tissue expression of IL-1&#946;; (B) Lung tissue expression of TNF-&#945;. The values for all the measurements are expressed as the means &#177; standard errors of the mean (SEMs), and comparisons were made with one-way ANOVA. BD, brain death; HS, hemorrhagic shock; CD, circulatory death. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62975/62975fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Study of Experimental Organ Donation Models for Lung Transplantation at JoVE.com

Study of Experimental Organ Donation Models for Lung Transplantation

The present study shows the establishment of three different lung donation models (post-brain death donation, post-circulatory death donation, and post-hemorrhagic shock donation). It compares the inflammatory processes and pathological disorders associated with these events.

Experimental models are important tools for understanding the etiological phenomena involved in various pathophysiological events. In this context, ...

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Nanyang Technological University

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Research

JoVE Journal

Medicine

1.6K Views.  Universidade de São Paulo. Experimentele orgaandonatiemodellen bootsen over het algemeen geïsoleerde situaties na van weefselbeschadiging die verband houden met het doodsproces en/of gebeurtenissen die verband houden met het behoud en de implantatie van het orgaan. Deze modellen zijn zeer nuttig bij de ontwikkeling van therapieën die het aantal donaties willen verhogen en bijgevolg de wegingslijst van potentiële ontvangers willen verlagen. Daarom is het belangrijk geworden om verschillende modellen te ontwikkelen, z...

Video: Study of Experimental Organ Donation Models for Lung Transplantation

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

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 ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

Bioengineering

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

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. ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

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 ...

A Simple Cuff Technique for Murine Left Lung Transplantation

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an efficient and highly reliable "cuff technique" of transplantation. This article describes a sophisticated and comprehensive method for orthotopic lung transplantation in a vascularized orthotopic lung model, representing the most physiologic and clinically relevant model of mouse lung transplantation to date. The transplantation process consists of two distinct stages: donor harvest and subsequent implantation into the recipient. The method has been successfully mastered, and with several months of sufficient training, a skilled practitioner can perform the procedure in approximately 90 min from skin-to-skin. Surprisingly, once individuals overcome the initial learning curve, the survival rate during the perioperative period approaches nearly 100%. The mouse model allows for the use of multiple commercially available transgenic and mutant strains of mice, enabling the study of tolerance and rejection. Additionally, the unique features of this model make it a valuable tool for investigating tumor biology and immunology.

The present protocol describes a cuff technique for a mouse left lung transplantation model. This technique has been developed over several years and has performed well, serving effectively in immunological research.

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an ...

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 ...

Study of Experimental Organ Donation Models for Lung Transplantation

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