Method Article

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

DOI:

10.3791/64954

⸱

April 21st, 2023

 ,  ,  ,  ,  ,  ,  , 
1Department of Medical Science, Chonnam National University Graduate School, 2Department of Thoracic and Cardiovascular Surgery, Chonnam National University Hospital and Medical School, 3Cardiovascular and Respiratory Research Team, Chonnam National University Hospital, 4Department of Internal Medicine, Chonnam National University Hospital and Medical School, 5Departments of Surgery, Chonnam National University Hospital and Medical School, 6Department of Pediatrics, Chonnam National University Children’s Hospital, and Medical School

In This Article

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Heart transplantation remains the sole viable therapy for refractory heart failure1,2,3,4. Despite a steady rise in the number of patients in need of heart transplantation, a proportional increase in the availability of donor organs has not been observed5. To address this issue, novel approaches for preserving donor hearts have been developed with the goal of improving the challenges and increasing the availability of donors6,7,8

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The ethical committee of the Laboratory Animal Research Center of Chonnam National University Hospital (approval no. CNU IACUC - H - 2022-36) approved all the animal experiments. Male Sprague-Dawley rats (350-450 g), used in this study received care in compliance with the guidelines for the care and use of the laboratory animals. The rats were housed in temperature-controlled rooms with a 12 h light-dark cycle, with standard food and water available.

1. Preparation

NOTE: A single experimenter can conduct all experimental procedures.

  1. Assemble the Langendorff apparatus, including the ox....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Figure 1 illustrates the experimental design used in a small animal model. Figure 2 displays the modified Langendorff perfusion apparatus, which includes a small animal oxygenator. The order of anastomosis for heterotopic abdominal implantation is presented in Figure 3.

Figure 4 shows the parameters used to assess the viability of the heart during ex situ perfusion, .......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Our focus in establishing this model was to replicate normothermic human heart transplantation. Non-ejecting models are the commonly preferred technique for preserving the donor heart in an ex situ environment16. While ejecting models offer many advantages in assessing cardiac function during ex situ perfusion17, they are not suitable for heterotopic transplantation models. In heterotopic transplantation, the implanted donor heart needs to overcome systoli.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have no conflicts of interest.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was supported by a grant B2021-0991 from the Chonnam National University Hospital Biomedical Research Institute and NRF-2020R1F1A1073921 from the National Research Foundation of Korea

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AES active evacuation systemSmiths medicalPC-6769-51AUtilize CO2 and excess isoflurane
Anesthesia machineSmiths medicalPC-8801-01AMixes isoflurane and oxyegn and delivers to animal
B20 patient monitorGE medical systemsB20to observe mean aortic pressure and temperature
Homeothermic Monitoring SystemHarvard apparatus55-7020To monitor and maintain animal's temperature
Micro-1 Rat oxygenatorDongguan Kewei medical instrumentsMicro-MOFor gas exchange in the langendorff circuit
Micropuncture introducer SetCOOK medicalG48007for delivering cardioplegic solution to the arch through the abdominal aorta
MicroscopeAmscopeMU1403For zooming surgical field (Recipient)
Surgical loupeSurgiTelL2S09For zooming surgical field (Donor)
Syringe pumpAMP allSP-8800To deliver cardioplegic solution
Transonic flow sensorTransonicME3PXL-M5Perfusion circuit flow sensor
Transonic tubing flow moduleTransonicTS410flow acquiring system
Watson - Marlow pumpsHarvard apparatus010.6131.DAOPeristaltic pump used for recirculate perfusate
WBC-1510AJEIO TECHE03056DHeating bath
Sprague-Dawley ratsSamtako Bio Korea Co., Ltd., Osan City Korea
Medications
BioHAnce Gel Eye DropsSENTRIX Animal carewet ointments for eye
CefazolinJW pharmaceuticalFor prophilaxis
CustodiolDR, FRANZ KOHLER CHEMIE GMBHFor heart harvesting
DiclofenacMyungmoon Pharm. Co. LtdFor pain control
HeparinJW pharmaceuticalAnticoagulant
InsulinJW pharmaceuticalhormon therapy
SalineJW pharmaceuticalFor hydration therapy

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Langmuur, S. J. J., et al. Normothermic ex-situ heart perfusion with the organ care system for cardiac transplantation: A meta-analysis. Transplantation. 106 (9), 1745-1753 (2022).
  2. Ardehali, A., et al.

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Erratum

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Formal Correction: Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation
Posted by JoVE Editors on 8/28/2023. Citeable Link.

An erratum was issued for: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation. The Protocol section was updated.

Section 4 of the Protocol was updated from:

4. Implantation

  1. Preparation of recipient
    1. Begin the recipient preparation 30 min before the cessation of ex situ perfusion.
    2. Anesthetize the recipient animal using the same method as mentioned in step 2.2.
    3. Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 °C.
    4. Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.
  2. Medications
    1. Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.
    2. Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.
    3. Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.
  3. Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.
  4. Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.
  5. Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15.
    NOTE: Do not separate the Abd. A. and IVC.
    1. Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.
    2. Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.
    3. Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.
    4. Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Be sure to switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia.
    5. Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.
    6. Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.
    7. Starting the anastomoses, placed the donor heart on the right side of the recipient's abdomen and attach the donor Asc. A to the recipient's Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.
    8. Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor's Asc. A with the recipient's Abd. A using a running 9-0 polypropylene suture.
    9. Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.
    10. Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.
  6. De-airing and de-clamping
    1. Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.
    2. Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.
    3. Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.
    4. Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline.
      NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat's body temperature is below 35 °C, the heart rhythm will normalize after the temperature reaches 36 °C.
  7. Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.

to:

4. Implantation

  1. Preparation of recipient
    1. Begin the recipient preparation 30 min before the cessation of ex situ perfusion.
    2. Anesthetize the recipient animal using the same method as mentioned in step 2.2.
    3. Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 °C.
    4. Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.
  2. Medications
    1. Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.
    2. Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.
    3. Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.
  3. Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.
  4. Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.
  5. Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15.
    NOTE: Do not separate the Abd. A. and IVC.
    1. Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.
    2. Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.
    3. Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.
    4. Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia. Ensure to switch on the heating pad after de-clamping the vessels to avoid hypothermia.
    5. Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.
    6. Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.
    7. Starting the anastomoses, placed the donor heart on the right side of the recipient's abdomen and attach the donor Asc. A to the recipient's Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.
    8. Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor's Asc. A with the recipient's Abd. A using a running 9-0 polypropylene suture.
    9. Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.
    10. Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.
  6. De-airing and de-clamping
    1. Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.
    2. Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.
    3. Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.
    4. Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline.
      NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat's body temperature is below 35 °C, the heart rhythm will normalize after the temperature reaches 36 °C.
  7. Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.
  8. After the surgery, place the anesthetized animal on a clean area over a heating pad until the body temperature reaches 37°C. 
    NOTE: Do not initiate the postoperative examinations till the body temperature reaches 37°C. Maintain anesthesia at 2-2.5% isoflurane until the end of the experiments.
  9. Monitor ECG of the transplanted donor heart for 3 h. Then, excise the heart under deep anesthesia for histological studies.
    NOTE: Confirm anesthesia depth via lack of pedal reflex before excising the heart. The surgical procedure and the ECG monitoring take less than 6 h. Diclofenac, administered perioperatively (step 4.2.3.), enables pain management for the entire duration of this procedure. The analgesia regimen can be adjusted per the institutional animal use guidelines.

Tags

Normothermic Ex Situ PerfusionHeterotopic Heart TransplantationRat Heart TransplantDonor Heart PreservationEx Situ Heart PerfusionLangendorff ApparatusMicrosurgical TechniquePerfusion CircuitCardioplegic SolutionImmune Response

Related Articles