A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Podsumowanie

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Streszczenie

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

Wprowadzenie

The number of patients experiencing ischemic diseases due to atherosclerosis is increasing worldwide¹. Despite the current advances in medical and surgical therapies for cardiovascular diseases, ischemic heart diseases, such as myocardial infarction, remain a major cause of morbidity and mortality². Furthermore, peripheral arterial diseases characterized by reduced blood flow to the limbs induces critical limb ischemia, wherein approximately 40% of the patients lose their legs within 6 months of diagnosis, and the mortality rate is up to 20%³.

Revascularization surgeries, such as coronary artery bypass grafting (CABG) and bypass surgery for peripheral arteries, are major therapeutic options for ischemic diseases. The purpose of these surgeries is to provide a new blood pathway to provide sufficient blood flow toward the distal site of the stenotic or occluded lesions of the atherosclerotic arteries. Although in situ arterial grafts, such as internal thoracic arteries for CABG, are preferred as the bypass grafts because of the expected longer patency, vein grafts, such as autologous saphenous veins, are commonly used because of the higher accessibility and availability⁴. The weak point of the vein grafts is the poor patency rate compared to that of artery grafts⁵ due to accelerated intimal hyperplasia when subjected to arterial pressure, which leads to vein graft disease⁶.

Vein graft disease develops through the following three steps: 1) thrombosis; 2) intimal hyperplasia; and 3) atherosclerosis⁷. In order to address vein graft disease, a lot of basic research has been conducted⁸. Thus far, no pharmacological strategy other than antiplatelet and lipid-lowering therapies are recommended for secondary prevention after coronary or peripheral revascularization surgeries in recent guidelines^9,10,11,12. Thus, to overcome vein graft disease, especially intimal hyperplasia, the establishment of a relevant experimental platform for further studies is required.

Intimal hyperplasia is an adaptive phenomenon that occurs in response to the change in the surroundings, where vascular smooth muscle cells (VSMCs) proliferate, accumulate, and generate extracellular matrix in the intima. Consequently, it presents a foundation for graft atheroma⁷. In the hyperplastic intima, VSMCs bear proliferation, and production rather than contraction, termed “phenotypic change”⁸. It is a key research target to control the phenotype of the VSMCs of the vein grafts to prevent vein graft disease, and numerous basic studies have been conducted on this topic⁸. However, a randomized controlled clinical study that aimed to achieve pharmacological control of the VSMC phenotype showed limited results¹³. Further, there are no standardized therapies to prevent intimal hyperplasia. More basic research, including animal model studies, is necessary.

To promote research in this field, it is crucial to establish an animal model that recapitulates vein grafts under arterial blood pressure, allowing a long-term, postoperative observation. Carrel et al. established a canine model of implantation of the external jugular vein into the carotid artery¹⁴. Therafter, a variety of vein grafts have been employed to investigate the physiological and pathological effects of alterations in arterial blood pressure, including the inferior vena cava engrafted into the thoracic or the abdominal aorta, or the saphenous vein engrafted into the femoral artery^15,16,17. These models were built in larger animals, such as pigs or dogs, that are suitable for mimicking a vein graft disease in a clinical case. However, the establishment of an animal model that can be prepared without special surgical techniques and at a lower cost would be ideal for researchers trying to develop a new therapeutic strategy for attenuating intimal hyperplasia through VSMC phenotype control in vivo. Initially, the interposition of the jugular vein into the carotid artery in a rabbit was introduced in the field of neurosurgery^18,19. Thereafter, it was applied to research on intimal hyperplasia^20,21. The initial model consists of venous interposition alone, thus saving time. Moreover, a subsequent study demonstrated that the preparation of a vein graft also affected the intimal hyperplasia²². Davies et al. evaluated the effect of balloon catheter injury on the intimal hyperplasia in a rabbit venous interposition model^23,24. Although balloon catheter injury in addition to vein interposition was more relevant to a clinical setting, a more reproducible model was also desired. Thus, Jiang et al. examined the impact of differential flow environments on intimal hyperplasia and established a distal branch ligation procedure as a reproducible model²⁵. However, they employed a cuff technique at the time of vein graft interposition that seems different from hand-sewn anastomosis in the clinical setting. In the present protocol, we report a reproducible, clinically relevant, and broadly available procedure for the preparation of a rabbit venous interposition model to assess intimal hyperplasia under arterial blood pressure.

Protokół

NOTE: All the surgical procedures performed on animals should be carried out in accordance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html) or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding.

1. Preparation of animals

1. Purchase male Japanese white rabbits (or rabbits with equivalent body size) weighing 2.7–3.0 kg.
2. Acclimate the rabbits for 1 week in a 12 hour light–dark cycle and feed a regular rabbit chow diet before the procedure.

2. Anesthesia and animal setting

NOTE: All the subsequent procedures must be performed under aseptic conditions. The surgical field and devices should be disinfected with 10% povidone-iodine solution, 70% alcohol, or a quaternary ammonium compound before use.

1. Anesthetize a rabbit with intravenous administration of pentobarbital sodium (25 mg/kg) via the auricular vein.
2. After ensuring that the rabbit has lost its strength, transfer it to an operating table, and set it in the supine position. Cover the nose and mouth with an anesthetic mask. Start the administration of general anesthesia with the inhalation of 0.7–1.0% isoflurane-oxygen mix.
   NOTE: If the rabbit starts to move, a temporal surge in isoflurane up to 2% will be effective.
3. Trim the fur on the neck and shoulder using an electric hair clipper. After disinfecting the surface by spraying 70% ethanol or another antiseptic solution, shave the rest of the hair in the cervical region with a razor. Disinfect the surgical field with 10% povidone-iodine and administer lidocaine hydrochloride (3 mg/kg) subcutaneously as local anesthesia.
   NOTE: Examine the repetitive movement of the trachea. Observe the pulsation of the jugular vein and the carotid artery. When the respiratory and pulsatile rates decrease, consider a temporary reduction in anesthesia administration. Monitoring the percutaneous oxygen saturation and pulse rate is also helpful.
   NOTE: The protocol can be paused here.

3. Harvest of the jugular vein

NOTE: Local anesthetics (such as lidocaine) should be used before making the skin incision.

1. Before skin incision, administer prophylactic enrofloxacin (5 mg/kg) subcutaneously. For analgesia, administer 0.05 mg/kg of buprenorphine subcutaneously twice a day for 3 days.
   NOTE: To avoid a drop in body temperature, a surgical scrub of the incision site can be used instead of spraying the animal's body with 70% ethanol.
2. Incise 50–60 mm of the cervical region with surgical scalpel longitudinally. Bluntly dissect the subcutaneous tissues and fascia to expose a 20–30 mm segment of the jugular vein. Ligate all the branches of the exposed vein with 4-0 silk sutures.
3. Place a 2-0 silk suture around the internal and external jugular veins to perform ligation immediately after incising the jugular vein in the next step.
4. Incise the venous wall (approximately 1 mm) of the distal side of the vein. Insert a 2-French balloon catheter from the cut toward the proximal side of the vein. Ligate the 2-0 silk suture at the distal sites of the jugular veins.
5. Inflate the balloon with 0.2 mL of air. Denude the intima of the vein using three passages of the catheter for endothelial exfoliation.
   NOTE: This procedure is considered equivalent to the distension of a saphenous vein graft in human revascularization surgeries, which causes endothelial exfoliation.
6. Ligate the proximal end of the vein. Cut the vein to harvest.
   NOTE: Carefully distinguish the distal and proximal end of the harvested vein, because the anastomosis to the artery should be performed in an inverted manner (i.e., the distal end of the vein should be anastomosed to the proximal end of the artery). For example, insertion of an intravenous catheter from a certain side would work as a marker.
7. For therapeutic manipulations for the harvested jugular vein, treat the harvested vein with methods designed for each research question (e.g., electroporation²⁶ or direct immersion with solutions²⁷ for transducing microRNAs into the veins).
   NOTE: For this protocol, the vein grafts were soaked in phosphate-buffered saline, control microRNA, and microRNA-145. The protocol can be paused here.

4. Interposing the carotid artery by the harvested jugular vein

1. Expose a 20–30 mm segment of the ipsilateral carotid artery. Separate the artery carefully from the vein and nerve nearby. Ligate all the branches of the exposed vein with 4-0 silk suture.
2. Administer heparin sodium intravenously (200 IU/kg). Wait for 3–4 min.
3. Clamp the proximal and distal ends of the artery with surgical clamps. Cut the artery in the middle, between the clamps. Inject normal saline into the incised carotid artery proximally and distally to distend the artery.
   NOTE: A rabbit carotid artery tends to shrink. Choose a well-distended site as an anastomosis site.
4. Anastomose the harvested vein to the artery in a reversed end-to-end fashion.
   1. Insert a 20 G intravenous catheter into the harvested vein from the distal to the proximal direction to keep the venous lumen open during the distal anastomosis.
   2. Anastomose the proximal end of the vein to the distal end of the artery using 8-0 polypropylene interrupted sutures. Place two anchor stiches at the site and the opposite site. Add stiches the upper side of the anastomosis line between the anchor stiches.
   3. Flip the artery and the vein graft upside down. Add stiches on the remaining part of the anastomosis line.
   4. Remove the intravenous catheter from the vein. Clamp the vein graft proximally and declamp the carotid artery distally. Ascertain that the vein graft is expanding gradually.
   5. Anastomose the distal end of the vein to the proximal end of artery using 8-0 polypropylene interrupted sutures. Declamp the artery to check the bleeding from the anastomosis sites. Add sutures for hemostasis, if required.
      NOTE: A gentle compression of the bleeding site with gauze and waiting may be enough for hemostasis. Check the immediate expansion and strong pulsation of the vein graft after the proximal declamping. If that is not observed, consider repeating steps 4.4.2–4.4.3
5. Ligate the internal carotid artery with a 4-0 silk suture to simulate a poor runoff condition and to facilitate intimal hyperplasia.
6. Clean the wound with saline. Close the wound using 3-0 polyglactin 910, layer by layer.

5. Postoperative procedures

1. End the anesthesia and remove the anesthesia mask after checking the spontaneous breathing of the animal. Check the conditions of the animal frequently until it recovers from anesthesia.
2. Keep the animal separated from other animals until respiratory function is fully restored. Support breathing manually, if necessary. Do not return the animal to a larger group until full recovery.
3. Check the food and water intake after recovery from anesthesia and provide appropriate nutritional support. Administer analgesics (e.g., buprenorphine 0.05–0.2 mg/kg subcutaneously 2x a day for 3 days) and check for signs of discomfort or pain.

Wyniki

Figure 1A shows a representative image of successful intimal hyperplasia at 2 weeks after venous interposition surgery (upper panel). The lower panel shows the therapeutic effects of microRNA-145-loaded poly(lactic-co-glycolic acid) nanoparticles that attenuated the intimal hyperplasia (lower panel). Figure 1B shows the comparison of intimal hyperplasia between the control group using phosphate buffered saline control (PBS), control microRNA (Cont-miR), and micr...

Dyskusje

The present protocol is designed to provide an experimental platform to test various molecular or genetic interventions for VSMCs to control the phenotype from the proliferative to the contractile state and subsequently attenuate the progression of venous intimal hyperplasia in vivo. Using this model, we successfully prepared intimal hyperplasia at 2 weeks after surgery (Figure 1A) and indicated the therapeutic potential of microRNA-145 to control the VSMC phenotype²⁶...

Materiały

Name	Company	Catalog Number	Comments
10% Povidone-iodine solution	Nakakita	872612	Surgical expendables
2-0 VICRYL Plus	Johnson and Johnson	VCP316H	Surgical expendables
4-0 Silk suture	Alfresa pharma	GA04SB	Surgical expendables
8-0 polypropylene suture	Ethicon	8741H	Surgical expendables
Cefazorin sodium	Nichi-Iko Pharmaceutical	6132401D3196	Antibiotics
Fogarty Catheter (2Fr)	Edwards Lifesciences LLC	E-060-2F	Surgical expendables
Heparin	Nipro	873334	Anticoagulant
Intravenous catheter (20G)	Terumo	SR-OT2051C	Surgical expendables
Isoflurane	Fujifilm	095-06573	Anesthesia
Lidocaine hydrochloride	MP Biomedicals	193917	Anesthesia
Pentobarbital sodium	Tokyo Chemical Industry	P0776	Anesthesia