Study of Experimental Organ Donation Models for Lung Transplantation

Summary

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.

Abstract

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.

Introduction

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 pharmacology^1,2,3. 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 challenges^2,4. 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 transplantation^5,6,7,8.

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 transplantation^6,7,8,9. 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's reflex^10,11. 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 mortality^10,11,12,13.

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 process^6,14.

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 growing^8,15,16,17.

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.

Protocol

Animal experiments complied with the Ethics Committee for Experimental Animals Use and Care of the Faculty of Medicine of the University of São Paulo (protocol number 112/16).

1. Animal grouping

1. 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.
2. 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.
3. 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.
4. 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.

2. Anesthesia and presurgical preparation

1. Place the rat in a closed chamber with 5% isoflurane for 1 - 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.
2. 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.
3. Remove fur from the regions of interest (head, neck, chest and abdomen). Then, using gauze, disinfect the surgical field and the animal's tail. Disinfection is performed with three alternating rounds of an alcoholic solution of chlorhexidine digluconate scrub.
4. Cut the tip of the animal'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 µL) 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).
5. Use a precision pipette to dilute the collected blood in 380 µL (1:20) of Turk'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.

3. Tracheostomy

1. 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 (1.5 cm incision). After the incision of the skin and subcutaneous tissue, dissect the cervical muscles until the trachea is exposed.
2. Place one 2-0 silk ligature beneath the trachea.
3. 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).
4. Insert the ventilation tube and fix it with prepared ligatures.
5. Connect the ventilation tube to the small-animal ventilation system.
6. Ventilate the rat with a tidal volume of 10 mL/kg, rate of 70 cycles/min, and PEEP of 3 cmH₂O.

4. Femoral artery and vein catheterization

1. Expose the femoral triangle through a small incision (1.5 cm) in the inguinal region. Identify and isolate the femoral vessels. For this procedure, use a stereomicroscope (3.2x magnification).
2. 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.
3. Insert the catheter through a small, pre-formed incision in the vessels. Fixate the cannula 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's venous network, thus preventing regurgitation of blood contents. Lubricate the cannula with heparin, avoiding the formation of thrombi and complications during mean arterial pressure (MAP) measurement.
4. 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's heart. Record the MAP every 10-min period.
5. Place the syringe catheter (3 mL) into the vein, aiming for hydration and exsanguination when necessary.

5. Hemorrhagic shock induction

1. Through venous access and with a heparinized syringe, remove small volumes of blood until MAP values of 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.
2. 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.
3. Put a source of heat nearby to avoid hypothermia.
   NOTE: Here, a heat lamp is used.
4. 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).

6. Circulatory death induction

1. To induce circulatory death, administer 150 mg/kg sodium thiopental through the venous line. Then turn off the ventilation system.
2. 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 °C) for 180 min.
3. 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.

7. Brain death induction

1. Place the rat in the prone position.
2. 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.
3. Insert the entire balloon catheter into the cranial cavity and ensure that the balloon is prefilled with saline (500 µL).
4. With the help of a syringe, rapidly inflate the catheter.
5. Confirm brain death by observing an abrupt MAP elevation (Cushing's reflex), the absence of reflexes, bilateral mydriasis, and apnea. After confirmation, discontinue anesthesia and keep the animal on mechanical ventilation for 360 min.
6. Place a source of heat nearby to avoid hypothermia.
7. 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).

Results

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 ± 6.1 vs. HS: 105.8 ± 2.3 mmHg; p=0.5; two-way ANOVA). After catheter insufflation, the BD group experi...

Discussion

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 content^4,18.

In ...

Materials

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