Name: a rabbit venous interposition model mimicking revascularization surgery using vein grafts to assess intimal hyperplasia under arterial blood pressure
Uploaded: 2020-05-15T13:30:00.000+00:00
Duration: 7 min 2 s
Description: Watch this Scientific Journal Video about A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure at JoVE.

This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (25462136).

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
<ol>
	<li>Purchase male Japanese white rabbits (or rabbits with equivalent body size) weighing 2.7&#8211;3.0 kg.</li>
	<li>Acclimate the rabbits for 1 week in a 12 hour light&#8211;dark cycle and feed a regular rabbit chow diet before the procedure.</li>
</ol>
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.
<ol>
	<li>Anesthetize a rabbit with intravenous administration of pentobarbital sodium (25 mg/kg) via the auricular vein.</li>
	<li>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&#8211;1.0% isoflurane-oxygen mix. 
	NOTE: If the rabbit starts to move, a temporal surge in isoflurane up to 2% will be effective.</li>
	<li>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&#160;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.</li>
</ol>
3. Harvest of the jugular vein
NOTE: Local anesthetics (such as lidocaine) should be used before making the skin incision.
<ol>
	<li>Before skin incision, administer prophylactic enrofloxacin (5 mg/kg) subcutaneously.&#160;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&#39;s body with 70% ethanol.</li>
	<li>Incise 50&#8211;60 mm of the cervical region with surgical scalpel&#160;longitudinally. Bluntly dissect the subcutaneous tissues and fascia to expose a 20&#8211;30 mm segment of the jugular vein. Ligate all the branches of the exposed vein with 4-0 silk sutures.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Interposing the carotid artery by the harvested jugular vein
<ol>
	<li>Expose a 20&#8211;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.</li>
	<li>Administer heparin sodium intravenously (200 IU/kg). Wait for 3&#8211;4 min.</li>
	<li>Clamp the proximal and distal ends of the artery with surgical&#160;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.</li>
	<li>Anastomose the harvested vein to the artery in a reversed end-to-end fashion.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Flip the artery and the vein graft upside down. Add stiches on the remaining part of the anastomosis line.</li>
		<li>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.</li>
		<li>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&#8211;4.4.3</li>
	</ol>
	</li>
	<li>Ligate the internal carotid artery with a 4-0 silk suture to simulate a poor runoff condition and to facilitate intimal hyperplasia.</li>
	<li>Clean the wound with saline. Close the wound using 3-0 polyglactin 910, layer by layer.</li>
</ol>
5. Postoperative procedures
<ol>
	<li>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.</li>
	<li>Keep the animal separated from other animals until respiratory function is fully restored. Support breathing manually, if necessary.&#160;Do not return the animal to a larger group until full recovery.</li>
	<li>Check the food and water intake after recovery from anesthesia and provide appropriate nutritional support. Administer analgesics (e.g., buprenorphine 0.05&#8211;0.2 mg/kg subcutaneously 2x a day for 3 days) and check for signs of discomfort or pain.</li>
</ol>

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The authors have nothing to disclose.

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 phenotype26,27, validating the present protocol as a model to further investigate the attenuation of intimal venous hyperplasia.
Our rabbit intimal hyperplasia model consists of the following three major steps: 1) catheter injury; 2) jugular vein interposition; and 3) distal branch ligation (Figure 3). Vein grafts injured with a balloon catheter passage were shown to be deendothelialized, and the intima was proved to be hyperplastic23,28. In this protocol, we consider the catheter-injured vein grafts as saphenous vein grafts that are often manually pressurized and dilated in clinical settings for procedures such as coronary or peripheral artery bypass graftings. Moreover, distal branch ligation promoted intimal hyperplasia due to the reduced flow rate25. In the reported low-flow model, where the internal carotid artery and all the external carotid arteries except the most inferior branch were ligated, the neointima was more enhanced compared to that in the high-flow model with no distal branch ligation. In contrast, the distal branch ligation in this protocol did not include external carotid artery ligation to simplify and minimize the procedures, and the additional external carotid artery ligation in the present protocol may result in greater neointimal progression.
Existing rabbit intimal hyperplasia models have been developed to pursue either reproducibility or clinical validity. The initial model comprised only jugular vein interposition21. Afterwards a few modifications were made, including catheter injury or distal branch ligation, to augment the extent of the intimal hyperplasia23,25. In contrast, we aimed to establish a reproducible model resembling a clinical case, where patients with diabetes mellitus or peripheral artery disease often present poor distal perfusion on the coronary or lower extremity circulation. Moreover, saphenous vein grafts are often manually distended through hydrostatic pressure that causes endothelial exfoliation29. Considering these multiple factors inducing intimal hyperplasia, we adopted the mixed venous interposition model combined with the two additional procedures, mimicking a revascularization surgery.
This procedure requires surgical dissections on relatively superficial layers of the body, and the surgical incision is limited to the cervical region, making it less invasive for animals, resulting in a higher survival rate, and facilitating long-term observation. The surgical anastomoses are performed at the surface level of the body. This enables different researchers, not only surgeons, to conduct the procedure. The only critical step would be the anastomosis procedure mentioned in step 4.4. Inaccurate stiches may result in a stenosis or occlusion at the anastomosis site. When 8-0 polypropylene sutures are created, use of surgical telescopes with a magnification of at least 2.5x are recommended. As described in another report that addressed the rabbit intimal hyperplasia model26, 10-0 sutures with the aid of 10x power microscopes would also be helpful. After perfecting our model, our patency rate of the implanted vein graft at 2 weeks postoperatively was 90.9%. Another advantage of our model is the relatively lower procedure cost compared to that of large animal experiments. This enables investigators to increase the experimental volume to perform a larger number of interventions.
This venous interposition model is more clinically relevant, easy to handle, and low-cost. It can be applied to larger animal models and used for clinical studies. Although porcine and canine CABG models have been developed30,31,32, they are technically demanding. A porcine jugular vein interpose model that consists of jugular vein interposition alone has been established33. Thus, it is possible that the two procedures of catheter injury and distal branch ligation would be additionally performed as being more valid.
Another possibility is applying our model to a mouse venous interposition model. A reported mouse venous interposition model adopted a technically demanding cuff technique to circumvent direct anastomosis34. In the field of vein graft disease, mouse experimental models as well as larger animal models have been commonly used35. These include intimal hyperplasia models and vein graft atheroma models36. Techniques involved in the preparation for intimal hyperplasia models in mice include carotid artery ligation37, wire injury38, vein interposition, and collar injury39. In the vein graft atheroma models, genetically modified mice are typically used in mouse experimental models, in contrast to larger animal models. A mixed venous interposition model with the carotid artery ligation in genetically modified mice would be more clinically relevant.
A limitation of the present procedure is that the flow pattern at the site of the interposed vein is not the same as those of the vein grafts in human surgeries. In particular, the blood flow of the vein grafts on CABG is provided in the diastolic phase, wherein the flow pattern is different from that in the systolic phase. Another limitation is that the establishment of animal models recapitulating the etiology of the ischemia mediated by atherosclerosis in humans is challenging. The use of genetically modified animals with impaired lipid metabolism or on a high-fat diet may help overcome this limitation in the future.

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 guidelines9,10,11,12. 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 &#8220;phenotypic change&#8221;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 artery15,16,17. 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 neurosurgery18,19. Thereafter, it was applied to research on intimal hyperplasia20,21. 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 model23,24. 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.

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

a rabbit venous interposition model mimicking revascularization surgery using vein grafts to assess intimal hyperplasia under arterial blood pressure

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.

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 microRNA-145 (miR-145) groups. MicroRNAs were loaded with poly(lactic-co-glycolic acid) nanoparticles. Treatment with microRNA-145 significantly attenuated intimal hyperplasia. Figure 2 shows immunostaining for Ki-67, a cell proliferative marker. Fewer Ki-67-positive cells are observed in the miR-145 group, indicating a phenotype change in the VSMCs from the immature proliferative state to the mature contractile state.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60931/60931fig1v2.jpg" /> 
Figure 1: Successful intimal hyperplasia of the veins.&#160;(A) Representative Elastica Van Gieson staining for samples treated with PBS (upper panel) and with microRNA-145-loaded poly(lactic-co-glycolic acid) nanoparticles (lower panel). Scale bars = 500 &#181;m. (B) Intimal area in the control PBS group, control microRNA group (Cont-miR), and microRNA-145 (miR-145) groups. MicroRNAs are loaded with poly(lactic-co-glycolic acid) nanoparticles. Statistical analyses were performed using one-way analysis of variance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60931/60931fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60931/60931fig2v2.jpg" /> 
Figure 2: Cell proliferation.&#160;Ki-67 staining (brown). Scale bars = 100 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60931/60931fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60931/60931fig3v2.jpg" /> 
Figure 3: Procedural schema.&#160;DNA = deoxyribonucleic acid; PLGA = poly(lactic-co-glycolic acid); RNA = ribonucleic acid. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60931/60931fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure at JoVE.

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

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.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

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5.7K Views.  Kyoto University. 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.

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

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Bioengineering

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

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.

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 ...

Rodent Venoplasty Balloon Model for Deep Vein Treatment Studies

Rodent Inferior Vena Cava Venoplasty Balloon Model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

Author Spotlight: Utilizing Venoplasty Balloon Model in Rodents to Simulate Surgical Interventions for Deep Veins

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic ...

Jugular Vein Graft Harvest From a Rabbit Model: A Surgical Procedure to Excise a Segment of Rabbit Jugular Vein for Ex Vivo Studies

Source: Nishio, H., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
In this video, we demonstrate a procedure for surgically harvesting a segment of the jugular vein from a rabbit model. The resected segment can be used as an autologous vein graft to study intimal hyperplasia that develops due to arterial blood pressure.

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...

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Leporine

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

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

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Chapters in this video

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Rodent Inferior Vena Cava Venoplasty Balloon Model

Jugular Vein Graft Harvest From a Rabbit Model: A Surgical Procedure to Excise a Segment of Rabbit Jugular Vein for Ex Vivo Studies

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model