Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Abstract

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.

Protocol

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.