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.
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.
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 lifespans8,9. 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 used13,14. 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.
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
2. Preparing the animals for surgery
3. Surgical procedures
4. Post-surgery care
5. Ultrasonic examination
6. Venous graft tissue collection
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 IU/L vs. 31.5 IU/L ± 2.58 IU/L), serum bilirubin (sham vs. VGD, 2.5 µmol/L ± 0.47 µmol/L vs. 4.5 µmol/L ± 0.14 µmol/L), total bilirubin (sham vs. VGD, 0.025 µmol/L ± 0.14 µmol/L vs. 0.92 µmol/L ± 0.33 µmol/L), and creatinine (sham vs. VGD, 92.75 µmol/L ± 4.15 µmol/L vs. 141.75 µmol/L ± 12.65 µmol/L).
Ultrasonic examination
All animals in the sham (n = 5) and VGD groups (n = 5) survived. The surgical procedures of CABG are shown in Figure 1. The mean operation time was 105 min ± 25 min (range: 90-160 min), and the mean intraoperative bleeding volume was 85 mL ± 35 mL (range: 50-200 mL). The influence of operation time is mainly the transition of the operator's proficiency from human to pig and has no special significance. The mean duration from after incision anastomosis to tracheal extubation was 17 min ± 5 min (range: 15-30 min). Ultrasound examination showed that the blood supply of the grafted vessel had partial regurgitation compared with the normal coronary artery, and the overall blood flow direction was generally normal (Figure 3). Pneumothorax, tamponade, infection, or other serious complications were not observed postoperatively. No significant difference was found in weight or BMI between the sham and VGD groups, 1 month postoperatively.
Ultrasonic examination was performed on the proximal end (Figure 3A,B), vascular cavity (Figure 3C,D), and distal end (Figure 3E,F) of the graft vessel. The retrograde flow was observed at the proximal and distal ends of the graft vessel; however, no blood extravasation was observed.
Pathologic changes in the veins
Every venous graft was evenly divided into three segments by length, and one section was selected from each segment for evaluation and classified according to the modified Proudilit classification for coronary artery stenosis18. Averaged values from the three sections were adopted as the results for the degree of occlusion. The specific classification was as follows: grade I = 0-point, normal without restenosis; grade II = 1-point, mild stenosis <30%; grade III = 2-points, stenosis between 30% and 50%; grade IV = 3-points, severe stenosis between 50% and 90%; grade V = 4-points, subtotal occlusion >90%; and grade VI = 5-points, total occlusion, with no blood flow to the venous graft. The modified Proudilit classification for coronary artery stenosis was adopted to assess the quantified results. The result for the sham group was 0.00 ± 0.00, indicating no vascular occlusion, while the result for the CABG group was 3.12 ± 1.22. Therefore, the difference was significant between the two groups (p < 0.05, Table 2).
Under the microscope, in the sham group, the tunica intima, tunica media, and the venous wall of the venous graft appeared normal. In the VGD group, the tunica intima and tunica medium of the venous grafts were significantly thickened 30 days after the CABG surgery. The tunica intima was ambiguously demarcated from the tunica media. The elastic layer of the tunica media disappeared (Figure 4). The lumen of the venous graft was filled with hyperplastic tissues (Figure 4). No significant change in vessel diameter was observed.
Figure 1: Outline of the procedure. (A–C) Pre-operation: Weigh the minipigs, check the performance of the defibrillator and ventilator, and connect the ventilation tube. (D–F) Anesthesia: Administer an intramuscular injection of anesthesia to the minipigs, fix the minipig on the operating table, fully expose the airway for tracheal intubation, connect the ventilator, and use inhalation anesthesia to maintain anesthesia. (G–I) During operation: Perform a preoperative ultrasound assessment of cardiac function in the minipigs and complete coronary artery bypass grafting through a left chest wall incision. (J–L) Post operation: Anastomose the wounds and pay attention to the post-operative care and feeding of the minipigs. Please click here to view a larger version of this figure.
Figure 2: The surgical procedure. (A) Cut the chest wall, (B) isolate the internal mammary vein, (C) remove the internal mammary vein, (D) perform heparin preconditioning, (E) suspend the pericardium, (F) perform myocardial ischemia reperfusion, (G) monitor the ECG changes, (H) block coronary blood flow, (I) anastomose the proximal end of the graft vessel, (J) distal coronary anastomosis site, (K) anastomoses distal to coronary arteries, (L) complete coronary artery bypass grafting. Please click here to view a larger version of this figure.
Figure 3: Ultrasonic examination. After the completion of coronary artery bypass grafting, the blood flow patency of the grafted vessel is assessed by ultrasound. (A,C,E) Normal coronary blood flow images. Continuous blood flow signals are seen at the (B) proximal, (D) middle, and (F) distal ends of the grafted vessels. Please click here to view a larger version of this figure.
Figure 4: Histological analysis. (A) The normal internal mammary vein pathological section showed clear vascular hierarchy and no lumen stenosis. (B,C) The pathology of the internal mammary vein 30 days after coronary artery transplantation showed that the intima of the vessel was thickened to varying degrees, and the lumen was obviously narrowed. Please click here to view a larger version of this figure.
Indicators | Sham Group (n=5) | Graft Group (n=5) |
ALT (IU/L) | 46 ±5.11 | 47.75±7.88 |
AST (IU/L) | 25.25 ±1.88 | 31.5±2.58* |
Total Protein (IU/L) | 63.12 ±.138 | 60.17±1.91 |
Albumin (IU/L) | 32.25 ±0.77 | 23.77±5.61 |
Globulin (g/L) | 30.87 ±.136 | 36.4±6.03 |
Serum Bilirubin (μmol/L) | 2.5 ±0.47 | 4.5±0.14* |
Total Bilirubin (μmol/L) | 0.025 ±0.14 | 0.92±0.33* |
Alkaline Phosphatase (IU/L) | 103 ±19.3 | 104±16.04 |
Glucosamine (mmol/L) | 4.44 ±0.36 | 5.96±0.42 |
Urea Nitrogen (mmol/L) | 2.46 ±0.17 | 2.89±0.65 |
Serum Creatinine μmol/L | 92.75 ±4.15 | 141.75±12.65* |
Total Cholesterol (mmol/L) | 2.37 ±0.12 | 2.16±0.06 |
Triglyceride (mmol/L) | 0.48 ±0.10 | 0.25±0.05 |
High Density Lipoprotein mmol/L | 1.05 ±0.07 | 1.03±0.07 |
Very Low Density Lipoprotein (mmol/L) | 1.43 ±0.06 | 1.29±0.04 |
Lactate Dehydrogenase (mmol/L) | 384.75 ±26.8 | 478.25±49.58* |
Table 1. Serum biochemical indicators. Statistical analysis software was used for analysis. Data were expressed as mean ± standard error (n = 5). Comparisons of measurement data were analyzed by the Student's t-test. A p–value less than 0.05 indicated statistical significance. *p < 0.05, CABG vs. sham.
Proudilit classification | ||||||
S. No. of sample | Score immediately after CABG surgery | Score 30 days after CABG surgery | ||||
1 | 0, 0, 0 | 2, 2, 2 | ||||
2 | 0, 0, 0 | 1, 2, 2 | ||||
3 | 0, 0, 0 | 2, 3, 2 | ||||
4 | 0, 0, 0 | 3, 2, 2 | ||||
5 | 0, 0, 0 | 2, 1, 2 |
Table 2. Statistical results of the graft occlusion degrees immediately after surgery and 30 days postoperatively. The modified Proudilit classification scale was used for vascular occlusion degree: grade I = 0-point, normal without restenosis; grade II = 1-point, mild stenosis <30%; grade III = 2-points, stenosis between 30% and 50%; grade IV = 3-points, severe stenosis between 50% and 90%; grade V = 4-points, subtotal occlusion >90%; and grade VI = 5-points, total occlusion, with no blood flow to the venous graft. The data include results from five venous grafts evenly divided into three sections by length.
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 operation group, the liver and kidney function of the animals in the operation group were damaged to a certain extent. Considering the occurrence of coronary graft disease, the weakening of myocardial contractility resulted in insufficient perfusion of peripheral tissues. The venous graft showed intimal hyperplasia and vascular remodeling 30 days after the CABG surgery (Figure 4). Fibrotic changes around the blood vessels are associated with wound healing, fibroblast proliferation occurs early in wound healing on day 1 to day 319, the production of active type I collagen and fibronectin occurs on day 4 to day 6, and cytoplasmic α-SM actin fibril aggregation occurs on day 7 to day 1419. Stress fibers imply the formation of myofibroblasts, which coincides with wound contraction20. It is unclear whether perivascular fibrosis affects surgical outcomes.
Here, we selected minipigs to establish the vein graft disease model. While small animals such as rats have been used to study the pathological mechanisms of VGD21, pigs are similar in size, anatomy, and physiology to humans and are, therefore, more suitable for studying the pathogenesis of human heart disease or as a tool for device development22. Internal mammary veins are also often selected as grafts clinically. Clinical studies from two independent groups found that internal mammary vein grafts have the characteristic of high incidence of vein graft lesions, and the same pathological changes were observed in our study (Figure 4)23,24. As in clinical practice, the selection of an appropriate surgical approach in animal surgery is critical to the success of the surgery; here, we referred to Hocum’s left thoracotomy11. We found that the left thoracotomy could clearly expose the operative field, the anatomy around the incision was easy to identify, and the amount of bleeding was low. In addition, compared with the median thoracotomy, the lateral thoracotomy does not require sawing of the sternum, so surgical stress can be reduced.
Anesthesia is crucial to the success of a surgical model. In this study, the protocol was modified from Kotani et al., with the combination of ketamine and diazepam used as anesthesia induction and isoflurane inhalation as maintenance anesthesia25. Additionally, a research group showed that intravenous drugs were also suitable for maintenance anesthesia26. Endotracheal intubation in pigs might be difficult for an animal surgical team. Compared with the human airway, the tracheal anatomy of pigs makes exposure of the glottis difficult27. Here, to better expose the glottis we pressed down the pig’s upper jaw to help expose the pig’s glottis (Figure 1D). On the other hand, the use of a direct laryngoscopy or a fiber-optic bronchoscopy will help visualize the glottis in endotracheal intubation28.
The pathological state of venous graft disease is mainly divided into three stages: 1) acute stage (within 1 month) thrombosis; 2) subacute stage (1-12 months) intimal hyperplasia; 3) late-stage (more than 12 months) formation of atherosclerosis, which is a cause of graft stenosis and occlusion29. Most of the changes in the acute phase of VGD are related to operational factors, and the atherosclerosis formed in the late stage is irreversible. The study of subacute endometrial thickening is very important for the pathogenesis, treatment, and prevention of VGD. It is also critical that the graft vessels chosen are different from the vertical vessels of the great saphenous vein. The internal mammary vein usually bears less hydrostatic pressure, and the pathological changes are faster after transplantation than for the great saphenous vein. In our model, typical intimal hyperplasia occluding the lumen of the grafted vessel was seen in the histological examination 30 days after surgery, and the same pathological changes have been observed in other clinical studies23,24. The modeling results of selecting the internal mammary vein in minipigs are stable in phenotype, the modeling time is short, and the degree of reduction of the pathological changes of VGD is high, which is conducive to the development of follow-up research.
The model also has some limitations. Some fine operations in the large animal modeling process, intraoperative monitoring of animal vital signs, and postoperative resuscitation all require certain practical experience, which requires professional surgeons and anesthesiologists to guide the training and greatly reduce the accidental mortality of animals. Large animal surgery requires specific experimental sites, professional staffing, and sufficient financial support, which may be a heavier burden for smaller institutes.
In conclusion, under the guidance of professionals, well-equipped laboratories can further study the pathological changes of VGD by establishing this minipig VGD model, which is of great significance for the treatment of VGD.
The authors have nothing to disclose.
The authors thank Guangdong Laboratory Animals Monitoring Institute for technical support, animal care, and sample collection. They also thank Shenzhen Mindray Bio-Medical Electronics Co., Ltd, for technical support in the ultrasonic examination. This work was supported by Guangdong Science and Technology Program, China, and Jinan University Central Universities Basic Scientific Research Business Expenses Project (2017A020215076, 2008A08003, and 21621409).
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 |