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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Y/N</td>
			<td>Clipping device</td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Shimadzu</td>
			<td>D314800051</td>
			<td>Analysis of the wet/dry weight ratio</td>
		</tr>
		<tr>
			<td>Barbiturate (Thiopental)</td>
			<td>Crist&#225;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>Blau Farmaceutica SA</td>
			<td>7000982-06</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Crist&#225;lia</td>
			<td>10,29,80,130</td>
			<td>Inhalation anesthesia</td>
		</tr>
		<tr>
			<td>Micropipette (1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>347765Z</td>
			<td>Handling of small- volume liquids</td>
		</tr>
		<tr>
			<td>Micropipette (20 &#181;L)</td>
			<td>Eppendorf</td>
			<td>H19385F</td>
			<td>Handling of small- volume liquids</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 experi...

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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632 Views.  Universidade de São Paulo. 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.

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 ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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 ...