Establishment and Evaluation of a Porcine Vein Graft Disease Model

Summary

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Abstract

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies.

To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated.

CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

Introduction

Although coronary heart disease mortality has declined significantly in recent years, half of middle-aged adults in the United States develop ischemic heart-related symptoms each year, and one-third of older adults die from coronary heart disease¹. Coronary artery bypass grafting (CABG) is an effective surgical modality to improve myocardial ischemia, and more importantly, it is an irreplaceable surgical modality for the treatment of multivessel coronary artery disease². Over time, however, vascular grafts develop inflammation, intimal hyperplasia, and progressive atherosclerosis, which is known to lead to vein graft failure or vein graft disease (VGD)³. In patients after CABG, if restenosis occurs, only the diseased blood vessel can be replaced in some cases². Older patients and added comorbidities make redoing coronary artery bypass grafting quite challenging. Delaying or controlling the pathological problems associated with grafted blood vessels is an urgent problem to be solved. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. Researchers have successfully established animal VGD models in small and large animals such as mice⁴, rats⁵, rabbits⁶, and pigs⁷. Compared with small animals, large animals such as pigs have anatomical structures and physiological characteristics similar to humans and have longer lifespans^8,9. Thus, large animals are more suitable for exploring long-term pathological changes in venous graft disease and for preclinical testing of drugs or devices. We and our collaborating team have successfully applied surgical techniques to establish a porcine heart failure model and described the cardiac pathological changes in this model¹⁰.

CABG surgery has been standardized in clinical practice, but when it is applied to the establishment of VGD animal models, the differences between species, the acquisition of animal equipment and facilities, animal surgical operations, and animal feeding and nursing are huge challenges for researchers. As in clinical practice, the approaches for CABG surgery used to establish VGD animal models include midline sternotomy¹¹ and left lateral thoracotomy¹². Midline sternotomy is more commonly used^13,14. However, this approach has high risks for both humans and animals. In the study reported by Thankam et al., two of the six pigs used for modeling died during surgery¹⁵. High model mortality increases study costs and affects the accuracy of results. A study showed earlier that a left chest wall incision was feasible to establish CABG-induced VGD in pigs¹¹. Here, this study aims to describe a step-by-step protocol to establish a reproducible surgery for a CABG-induced VGD model in minipigs and to evaluate the phenotype of this model. The experimental protocol was jointly designed by the cardiac surgery and anesthesia teams. The surgical approach for the left third intercostal space was determined according to the cadavers of other minipigs in the laboratory before surgery, and the anesthesia method was performed according to the method used at the center¹⁶. Blood biochemical tests, ultrasonic examination, and histology examination were conducted to evaluate animal models.

Protocol

The procedures for the care and use of laboratory animals were approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th Ed., 2011, National Research Council, USA). The surgical procedure is shown in Figure 1.

1. Preoperative preparation of animals

1. Randomly divide 10 3-month-old male minipigs weighing 30-35 kg into the sham group (n = 5) and VGD group (n = 5).
2. Evaluate the preoperative and postoperative health conditions of the pigs using the body mass index (BMI). Calculate the BMI as follows:
   BMI = body weight (kg)/(body length [cm] × body length [cm])
   NOTE: Body length is measured from the pig's nose to the base of the tail.
3. Fast the animals for 12 h before surgery to avoid aspiration after anesthesia. Prepare anesthesia appliances and surgical instruments, including an anesthesia machine, gas, anesthetic drugs, an anesthesia pipeline, a special laryngoscope, and surgical instruments, a rib retainer, sutures, a thyroid retractor, surgical forceps, etc. Sterilize all instruments to be used in the surgery.

2. Preparing the animals for surgery

1. Weigh the animals and calculate the anesthetic dose. Administer intramuscularly the anesthetic mixture comprising 2 mg/kg of 1:1 Tiletamine and Zolazepam, 0.2 mg/kg diazepam, and 0.02 mg/kg atropine¹⁷. Use fentanyl (50 mg/kg) for intraoperative pain relief³⁰.
2. Ensure that a proper anesthetic plane is achieved and insert an indwelling venous catheter (20G) into the marginal ear vein to establish ear access. Transfer the pig onto the operation table and place it in the supine position. Immobilize the limbs with bandages and elevate the head with a sterile drape.
   NOTE: The state of anesthesia was monitored by central fixation of the eyeball, miosis, loss of pupillary reflex, and loss of pain reflex.  The heart rate and blood pressure were maintained at a lower level than the baseline. The surgeon should monitor HR, BP, and other parameters under paralysis and increase the anesthetic dose if HR increases > 20% above baseline.
3. Expose the epiglottis and glottis using a veterinary laryngoscope. Perform tracheal intubation with a 7.0-7.5Fr tube and connect it to the anesthesia breathing circuit.
   NOTE: The ventilator is used for continuous positive pressure ventilation with tidal volume 280 mL, inspiratory/expiratory ratio 1:2, respiratory rate 20 times/min, and positive end-expiratory pressure (5 cm H₂O).
4. Intravenously inject vecuronium bromide (0.1 mg/kg) to relax the muscles during the surgical procedures and use 2% isoflurane to maintain anesthesia at a respiratory rate of 16-20 bpm and a tidal volume of 10 mL/kg.
   NOTE: Vecuronium is given to ensure adequate depth of anesthesia in paralyzed animals, especially since the dose of induction drug and isoflurane is on the lower end of recommended.
5. Use vet ointment on the pig's eyes to prevent dryness while under anesthesia. Use electric blankets to maintain the pig's body temperature at 38 °C ± 5 °C.
6. Use an electrocardiogram to monitor heart rate, blood oxygen levels, and body temperature.

3. Surgical procedures

1. Shave the left chest wall and apply three alternating rounds of 0.7% iodine and 75% alcohol to aseptically prepare the surgical area up to the left mandibular angle, down to the umbilical cord, left to the posterior axillary line, and right to the axillary front. Place a sterile surgical drape around the surgical area.
2. Make a 7-10 cm transverse incision with an electric knife in the third left intercostal space and separate the subcutaneous tissues layer by layer (Figure 2A). Remove a 5-6 cm segment of the third rib with bone scissors and expose the internal mammary vein by a retractor after exposing the third rib-sternal joint (Figure 2B).
3. Locate the internal mammary vein along with the left internal mammary artery on the left side of the sternum. Perform a blunt dissection of the internal mammary vein with vascular forceps.
4. Perform hemostasis by electrocoagulation of the branches of the left internal mammary vein with an electric knife. If hemostasis is incomplete, use cotton thread ligation for hemostasis. Ligate and mark the two ends of the vein while it is being harvested (Figure 2C).
5. Prepare heparin normal saline by adding 2 mL of heparin sodium solution and 98 mL of normal saline. After removing the vein, inject heparin normal saline into the vein for pretreatment (Figure 2D). Then, put the vein into normal saline and keep it for backup.
6. Make a similar incision as described above and remove the internal mammary vein in the sham group. Open the pericardium, then close the chest wall in the sham group. Use the internal mammary vein of the sham group for pathological control without coronary artery bypass grafting.
7. Make a ~7 cm incision with an electric knife on the pericardium to expose the right coronary artery trunk. Suspend the pericardium and sew on the skin on the ipsilateral side with the 1-0 surgical sutures (Figure 2E). Separate the right coronary artery trunk from the surrounding tissues (Figure 2E).
8. Bypass the blocking band under the proximal end of the isolated right coronary artery near the aorta with a wire hook and treat the myocardium with three cycles of 2 min ischemia and 5 min reperfusion by tightening and relaxing the blocking band (Figure 2F). Monitor the heart's electrical activity with the electrocardiogram monitor during the ischemia/reperfusion preconditioning (Figure 2G).
   NOTE: When the right coronary artery is blocked, the electrocardiogram shows an increased heart rate and ST-segment elevation.
9. Tighten the band to block the right coronary blood flow. Cut the epicardium covering the blood vessels. Expose the coronary artery wall, and cut longitudinally with the tip of a surgical blade against the center of the anterior wall of the blood vessels.
10. After cutting the lumen, enlarge the incision with scissors and place a coronary shunt. Insert one end of the shunt with a coil into the distal coronary artery through the tear. Shunt the blood in the coronary arteries into the hollow coronary shunt to ensure a clear operative field (Figure 2H).
11. Perform an end-to-side continuous suture between the internal mammary vein and the right coronary trunk with the 7-0 polypropylene suture (Figure 2I). In the middle of the ascending aorta, occlude the left anterolateral wall of the ascending aorta with a semi-occlusion clamp.
12. Use a surgical blade to make a small incision in the aortic wall where the adventitia has been cut, insert the head end of the sliding shaft at the head end of the punch into the aortic cavity through this incision, contract the sliding shaft outward, and the circular knife above it cuts off a piece of the arterial wall. The tissue block cut out by the punch is about 3 mm in diameter (Figure 2J).
13. Pull out the shunt. Perform an end-to-side continuous suture between the internal mammary vein and the the aortic wall with the 6-0 polypropylene suture (Figure 2K). Open the semi-occlusion clamp.
14. Record the bypass flow of the right coronary artery trunk proximal to the anastomosis site using ultrasound. Monitor the heart's electrical activity using the electrocardiogram (Figure 2L).
15. Indwell a temporary drainage tube (Fr: 16) into the chest cavity to allow the blood and fluids to drain. Sew the pericardium incision using a 1-0 cotton thread and close the chest layer by layer (from the inside to the outside: Pleura layer, muscle layer, subcutaneous tissue layer, skin layer) while placing penicillin (about 0.5 g) powder onto each layer. Remove the drainage tube after sewing the skin incision using a 1-0 cotton thread.

4. Post-surgery care

1. Remove the endotracheal tube after the animals returned to spontaneous breathing. The anesthesiologist should assess the animal’s vitals (e.g., respiratory rate, heart rate, oxygen saturation, etc.) and remove the ECG after the animals wake up and return to spontaneous activity. Send the animals back to the feeding room and place the sham animals in another pen in the breeding room. Keep the animals warm with an electric blanket. Observe the animals every hour after surgery (at least 4 times).
2. Feed the animal the day after surgery. Add aspirin (200 mg) 2x daily for 7 days to the animal feed to prevent post-operative thrombosis and reduce wound pain.
   NOTE: Avoid feeding animals on the day of surgery to prevent aspiration. 
3. Administer the animal with an intramuscular injection of penicillin 1x daily for 7 consecutive days to prevent post-operative infection (14,000 units per kg).

5. Ultrasonic examination

1. After CABG surgery, use a sterile ultrasonic probe sleeve to wrap the high-frequency linear array probe. Place the probe on the surface of the venous graft.
2. Display the outline of the graft in the two-dimensional ultrasound mode, then shift to the color Doppler mode to detect blood flow in the graft.

6. Venous graft tissue collection

1. Collect 10 mL of blood sample from the venous circuit of the ear vein for biochemical testing. (Table 1). Centrifuge the blood sample at 1,000 x g for 5 min and perform biochemical tests with an automatic biochemical analyzer.
2. Anesthetize the animal as described earlier. Upon confirmation of anesthesia depth, inject 10% potassium chloride 0.5mL/kg body weight from the ear marginal vein or forelimb vein. Then, make a 10 cm median sternal incision with an electric knife to harvest the vein graft 30 days after surgery. Fix the body position as in step 2.2., and after sterilization and placement of a drape, make a median sternum incision to split the sternum. During the separation, avoid the main blood vessels and the heart, and separate the grafted blood vessels layer by layer.
3. Quickly cut off the large blood vessels connecting to the heart, place the heart and ascending aorta on ice chips, and remove the graft vascular bridge, the connected aorta, and the right coronary artery. Rinse all the samples with normal saline at 4 °C.
4. Take the entire graft vessel of about 3-4 cm in size, divide it into 4-5 equal parts, and transfer to cryopreservation tubes. Quickly put the tubes into liquid nitrogen to flash freeze and move to a −80 °C ultra-low temperature freezer for storage.
5. For analysis, rinse the graft with ice-cold 0.9% saline and fix it in 4% paraformaldehyde solution. Maintain a ratio of tissue block size to fixative solution of 1:10 and fix the tissue for more than 12 h.
6. Stain the sections in 50 mL of aqueous solution of hematoxylin for 3 min. Separate the sections by washing with 50 mL of 0.5% hydrochloric acid ethanol and 50 mL of 0.2% ammonia water for 10 s each.
7. Rinse with running water for 1 h and then clean in distilled water by soaking for 3 min. Dehydrate in 70% and 90% ethanol for 10 min each. Place in 50 mL of 0.5% alcohol eosin staining solution for 2-3 min.
8. Dehydrate the stained sections with pure ethanol for 10 min and then soak in pure xylene for 10 min to make the samples transparent. Drip the transparent sections with neutral glue and cover with a coverslip. Observe pathological sections under a light microscope at 40x magnification.

Results

BMI and serum biochemical indices
The BMI between the sham and VGD groups was not significantly different (sham vs. VGD, 22.05 kg/cm² ± 0.46 kg/cm² vs. 21.14 kg/cm² ± 0.39 kg/cm², p = 0.46). The serum biochemical results are listed in Table 1. Statistically significant changes between the groups were found in four biochemical indexes, including aspartate aminotransferase (AST, sham vs. VGD, 25.25 IU/L ± 1.88 I...

Discussion

In this study, we described in detail the protocol for animal selection, instrument preparation, surgical procedures, and post-operative evaluation when developing a CABG-induced VGD model. We performed ultrasonic examination of the venous graft before and after CABG surgery and histological examination of the graft 30 days after the surgery. The blood flow in the internal mammary vein was normal before the CABG surgery, while retrograde flow was observed in the graft of the internal mammary vein. Compared with the sham ...

Materials

Name	Company	Catalog Number	Comments
Aortic Punch	Medtronic Inc. , America	3.0mm, 3.5mm, 4.0mm	Used for proximal coronary bridge anastomosis
Automatic biochemical analyzer	IDEXX Laboratories, Inc. America	Catalyst One
Cardiac coronary artery bypass grafting instrument kit	LANDANGER, France
Cardiogram monitor	Shenzhen Mindray Bio-Medical Electronics Co, Ltd	MEC-1000
Coronary Shunt	AXIUS	OF-1500, OF-2500, OF-3000	The product temporarily blocks the coronary artery during arteriotomy to reduce the amount of bleeding in the surgical field and provide blood flow to the distal end during anastomosis.  The Axius shunt plug is not an implant and should be removed prior to completion of the anastomosis.
Defibrillator	MEDIANA	Mediana D500
Diazepam	Nanguo pharmaceutical Co. LTD, Guangdong, China	H37023039	Narcotic inducer
Disposable manual electric knife	Covidien, America	E2516H
Electric negative pressure suction machine	Shanghai Baojia Medical Instrument Co, Ltd	YX932D
Esmolol	Guangzhou Wanzheng Pharmaceutical Co. LTD	H20055990	Emergency drugs
Ice machine	Local suppliers, Guangzhou, China
Lidocaine	Chengdu First Pharmaceutical Co. LTD	H51021662	Emergency drugs
Luxtec headlight system	Luxtec, America	AX-1375-BIF	Used for lighting fine parts during operation
Medical operation magnifier (glasses)	Germany Lista co, LTD	SuperVu Galilean 3.5×	Used for fine site operation during operation
Multi-function high-frequency electrotome	Shanghai Hutong Electronics Co, Ltd	GD350-B
Nitrogen canister	Local suppliers, Guangzhou, China
Nonabsorbable surgical suture (polypropylene suture)	Johnson & Johnson, America	6-0, 7-0	Used to suture blood vessels.
Nonabsorbable suture (cotton thread)	Covidien, America	1-0	Used for skin and muscle tissue tugging
Open heart surgery instrument kit	Shanghai Medical Instrument (Group) Co., LTD
Propofol injection	Xi 'an Libang Pharmaceutical Co. LTD	H19990282	Anesthetic sedative
Refrigerator	Local suppliers, Guangzhou, China
Respiratory anesthesia machine for animal	Shenzhen Reward Life Technology Co, Ltd, China	R620-S1
Semi-occlusion clamp	Xinhua Surgical Instrument Co., Ltd.	ZL1701RB	Temporarily cut off the aortic flow
vecuronium bromide	Richter, Hungary	JX20090127	Muscle relaxant
Veterinary ultrasound system	Royal Philips, Netherlands	CX50
Zoletil	Virbac, France	Zoletil 50	Animal narcotic