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

Summary

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.

Abstract

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.

Introduction

Most tissues and organs in the human body are dependent on a functional blood vessel network that supplies nutrients, exchanges gases and removes waste products. Malfunction of this system caused by local or systemic vascular problems can lead to a multitude of severe diseases. Moreover, in research areas such as tissue engineering or regenerative medicine, a functional blood vessel network within artificially generated tissues or transplanted organs is indispensable for successful clinical application.

For decades researchers have been investigating the exact mechanisms involved in the growing vasculature to gain deeper insight into pathological situations in order to find novel therapeutic interventions and provide better prevention of vascular disorders. In the first step, basic processes such as cell-cell interactions or the effect of molecules on cells of the vascular system are usually investigated by in vitro 2D or 3D experiments. Traditional 2D models are easy to perform, are well established and have contributed greatly to a better understanding of these processes. For the first time in 1980, Folkman et al. reported in vitro angiogenesis seeding of capillary endothelial cells on gelatin coated plates¹. This immediately gave way to publication of a multitude of further 2D angiogenesis experiments on endothelial cell tube formation assay², migration assay³ and the co-culturing of different cell types⁴, as well as others. These assays are still used today and accepted as standard in vitro methods.

However, this experimental setup is not always appropriate for the study of in vivo cell behavior since most cell types require a 3D environment to form relevant physiological tissue structures⁵. It could be shown that the architecture of the 3D matrix is decisive for capillary morphogenesis⁶ and that cell-extra cellular matrix (ECM) interactions and 3D culture conditions regulate important factors involved in tumor angiogenesis⁷. The 3D matrix provides complex mechanical inputs, can bind effector proteins and establish tissue-scale solute concentration gradients. Moreover, it is considered necessary in order to imitate in vivo morphogenetic and remodeling steps in complex tissues⁵. In these systems, both angiogenesis and vasculogenesis can be studied. While angiogenesis describes the sprouting of capillaries from preexisting blood vessels⁸, vasculogenesis refers to the de novo formation of blood vessels through endothelial cells or their progenitors^9,10. Maturation of vessels is described in a process called 'arteriogenesis' via recruitment of smooth muscle cells¹¹. A typical angiogenic in vitro model is the sprouting of endothelial cells from existing monolayers seeded as a monolayer on gel surfaces, on surface of microspheres embedded within a gel or by building endothelial cell spheroids¹². In vasculogenic models single endothelial cells are entrapped in a 3D gel. They interact with adjacent endothelial cells to form vascular structures and networks de novo, typically in combination with supportive cells¹².

However, even complex 3D in vitro models cannot mimic in vivo settings completely given the multitude of cell-cell and cell-ECM interactions¹³. Substances with high in vitro activity do not automatically show the same effects in vivo and vice versa¹⁴. For a comprehensive analysis of vascularization processes there is an urgent need to develop in vivo models that better simulate the situation in the body. A large range of in vivo angiogenesis assays are described in the literature, including the chick chorioallantoic membrane assay (CAM), the zebrafish model, the corneal angiogenesis assay, the dorsal air sac model, the dorsal skinfold chamber, the subcutaneous tumor models¹⁴. However, these assays are often associated with limitations, such as rapid morphological changes, problems in distinguishing new capillaries from already existing ones in the CAM assay, or the limited space in the corneal angiogenesis assay¹⁵. Furthermore, non-mammalian systems are used (e.g., the zebrafish model¹⁶), which leads to problems in xenotransplantation¹⁷. In the subcutaneous tumor model, angiogenesis originating only from the tumor itself cannot be analyzed since the adjacent tissue greatly contributes to the vascularization process. Moreover, the surrounding tissue can have a decisive role in shaping the tumor microenvironment¹⁸.

Not only for studying angiogenesis or vasculogenesis is there a strong need for a standardized and well-characterized in vivo model but also for studying different vascularization strategies in tissue engineering and regenerative medicine. Today, the generation of complex artificial organs or tissues is possible both in vitro and in vivo. 3D bioprinting provides an on-demand fabrication technique for generating complex 3D functional living tissues¹⁹. Furthermore, bioreactors can be used for generating tissues²⁰ or even the own body can be used as bioreactor²¹. However, the main barrier to successful application of artificially generated tissues is the lack of vascularization within the engineered constructs. Immediate connection to the host's vasculature after transplantation is a major prerequisite for survival, especially in the case of large-scale artificial tissues or organs.

Different in vitro or in vivo prevascularization strategies were developed to establish a functional microvasculature in constructs prior to implantation²². The implantation of a scaffold with in vitro preformed engineered capillaries onto the dorsal skin of mice led to rapid anastomosis of the mice vasculature within a day²³. In contrast, a spheroid co-culture consisting of human mesenchymal stem cells and human umbilical vein endothelial cells assembled into a three-dimensional prevascular network developed further after in vivo implantation. However, anastomosis with the host vasculature was limited²⁴. Above all, in poorly vascularized defects, such as necrotic or irradiated areas, this so-called extrinsic vascularization — the ingrowth of vessels from the surrounding area into the scaffold — often fails. Intrinsic vascularization, on the other hand, is based on a vascular axis as a source of new capillaries sprouting into the scaffold²⁵. Using the axial vascularization approach, the engineered tissue can be transplanted with its vascular axis and connected to local vessels at the recipient site. Immediately after transplantation, the tissue is adequately supported by oxygen and nutrients, which creates the right conditions for optimal integration.

Due to the limited availability of models for investigating in vivo angiogenesis and in recognition of the growing importance of generating axially vascularized tissue, we developed the microsurgical approach of Erol and Spira further to generate an arteriovenous (AV) loop in the animal model²⁶. The use of a completely closed implantation chamber makes this method very well suited to study blood vessel formation under "controlled", well characterized in vivo conditions (Figure 1). This model is not only useful for the investigation of angiogenesis but is also optimally suited for the axial vascularization of scaffolds for tissue engineering purposes.

Protocol

The Animal Care Committee of the Friedrich-Alexander University of Erlangen-Nürnberg (FAU) and the Government of Middle Franconia, Germany, approved all the experiments. For the experiments, male Lewis rats with a body weight of 300 - 350 g were used.

1. The Arteriovenous Loop Model in the Rat

1. Implantation Procedure (Figure 2)
   1. For anesthesia use a special plastic box that is connected via tube to the isoflurane vaporizer and closed by a lid. Turn on supply gas and flow meter between 0.8 - 1.5 L/min.
   2. Place the rat in the induction plastic box and seal the top. Turn on isoflurane vaporizer to 5%.
   3. Carefully observe the rat during induction of anesthesia. After 3 - 4 min, the rat will be anesthetized.
   4. Confirm proper anesthesia: loss of righting reflex, loss of palpebral reflex, withdrawal reflex, positive corneal reflex.
   5. Remove the rat from the box and check its weight for calculation of drugs. Apply eye ointment to prevent dryness while under anesthesia.
   6. Administer pain medication and antibiotics (e.g., 7.5 mg/kg enrofloxacin subcutaneously (s. c.), 12.5 mg/kg tramadol and 100 mg/kg metamizole both intravenously (i. v.)). Administer weight-adapted crystalloids during operation (e.g., 30 ml/kg s. c.).
   7. Place the rat on its back on a warming plate at 37 °C under anesthesia with 1 - 2% isoflurane inhalation administered via mask.
   8. Monitor anesthesia properly and increase isoflurane if anesthesia level is too low (movement of the rat, response to pain, jaw tone, no loss of reflexes (see 1.1.4.), heart rate increasing). Be careful not to overdose isoflurane (loss of corneal reflexes, high heart rate, decrease in oxygen saturation). Use a special pulse oximetry for small animals for checking the oxygen saturation (95 - 100%) and heart rate (250 - 450/min) of the rat. During the operation monitor temperature of the rat (36 - 40 °C) and adjust temperature of the warming plate if necessary.
   9. Shave the inner sides of the hind limbs with an electric razor and disinfect the area with antiseptics. Spread the hind limbs and fix them with adhesive tape.
   10. Lay the rat under a surgical microscope and cover the rat with sterile draping. Ensure that the whole operation procedure is performed under sterile conditions.
   11. Open the skin in the middle of the left thigh with a longitudinal incision from the upper knee to the groin using a scalpel (No. 10).
   12. Cut the subcutaneous tissue and fascia in layers of approximately 3 cm in length using dissecting scissors and microforceps until the femoral vascular bundle is exposed from the pelvic artery in the groin to the bifurcation of the femoral artery in the knee.
   13. Separate the vessels and remove the adventitia using adventitia scissors and microforceps.
   14. Coagulate the side branches using electric coagulation. Cover the operation field with a damp compress.
   15. Open the skin on the right side as described for the left side, 1.1.11 - 1.1.14.
   16. For harvesting the venous graft, ligate the right femoral vein by electric coagulation on the proximal and distal ends at a distance of 1 - 1.5 cm.
   17. Remove the venous graft with microforceps and flush the venous graft with a heparin solution (50 IU/ml in 0.9% sodium chloride solution) using an irrigation cannula and transfer it to the left thigh. Cover the operation field at the right side with a damp compress.
   18. Ligate the femoral vein on the left side proximally at the inguinal region with a microvessel clamp. Coagulate the femoral vein distally by electric coagulation, at the upper knee before branching, at a distance of about 2 cm.
   19. For anastomosis connect the proximal end of the venous graft with the proximal end of the vein by end-to-end anastomosis with an 11-0 suture. Use about 8 interrupted sutures. Begin with placing the first two sutures at the 12 o’clock and 6 o’clock positions. Then, put in 2 to 3 more sutures between these points on the front side and then put 2 to 3 more sutures into the back side.
   20. Ligate the femoral artery in the same way described for the femoral vein (1.1.18.). Make sure the loop vessels are not twisted. Anastomose the distal end of the venous graft with the proximal end of the artery as described for the femoral vein (steps 1.1.19.).
   21. Administer 25 IU heparin intravenously. Make again sure the loop vessels are not twisted, Remove the clamps and check for leakage and patency of the loop for about 5 min.
   22. Dribble papaverine (e.g., 4 mg/ml) on the vessels to prevent vascular spasms. If there is patency, the loop expands and the pulse of the artery can be observed.
   23. Prefill the implantation chamber with the first half (about 500 µl) of the matrix (e.g., a hydrogel or a bone matrix with or without cells). Embed the loop into the implantation chamber.
   24. Fill the implantation chamber with the second half of the matrix to a total volume of 1,000 µl. Seal the chamber with the chamber lid.
   25. Fix the implantation chamber on the thigh with a non-absorbable 6-0 suture. Fix the chamber lid with a non-absorbable 6-0 suture. Stop possible bleeding with electric coagulation.
   26. Close the skin with absorbable 4-0 sutures. Cover the wound with aluminum spray.
   27. Administer antibiotics and analgesics (e.g., 7.5 mg/kg enrofloxacin s. c., tramadol 12.5 mg/kg per os (p. o.) (through drinking water) for 3 - 5 days and afterwards depending on the behavior of the rat).
   28. Turn the vaporizer off and allow the rat to breathe supply gas until it begins to awaken.
   29. Place the rat in a box with thermal support and observe it carefully until fully recovered.
   30. Do not leave the rat unattended until it has regained sufficient consciousness to maintain sternal recumbency.
   31. Do not return the rat to the company of other animals until fully recovered.
2. Explantation Procedure
   1. Perform the explantation after a short-time or long-time implantation (according to the study design, for examples please see References 27-32). Anesthetize the rat according to steps 1.1.1.-1.1.9.
   2. Open the skin of the abdomen with a scalpel (No. 10) and move the intestines aside with a compress. Expose the abdominal aorta and the vena cava caudalis using cotton swabs.
   3. Cannulate the abdominal aorta (e.g., 24 gauge plastic cannula) and cut the vena cava caudalis with dissecting scissors.
   4. Flush the abdominal aorta with 0.9% sodium chloride solution containing 100 IU/ml heparin until the leaking fluid is clear. Perfuse the aorta with 30 ml perfusion solution (e.g., contrast agent or India ink).
   5. Euthanize the rat by injection of a lethal dose of embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution (0.1 - 0.2 ml/kg) in deep anesthesia.
   6. Ligate the abdominal aorta and vena cava caudalis with a non-absorbable 4-0 suture.
   7. Close the open wound area with 1 - 2 clamps. Store the rat for 24 hr at 4 °C (curing of the perfusion solution).
   8. Place the rat on its back. Spread the hind limbs and fix them with adhesive tape.
   9. Open the skin above the chamber with a longitudinal incision with a scalpel (No. 10).
   10. Remove the connective tissue from the chamber and the loop pedicle using dissecting scissors and microforceps. Cut the loop pedicle at a distance of 1 cm from the chamber opening with dissecting scissors and remove the chamber.
   11. Open the lid of the chamber and carefully remove the construct from the chamber with forceps and fix it in 4% buffered formalin solution for 24 hr at room temperature. Afterwards use the construct for 3D micro-computed tomography or paraffin-embedded for histological analysis³⁰.

Results

Tissue Engineering
For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat AV loop model^27,28,33,34. Vascularization could perfectly be demonstrated by 3D micro-computed tomography (micro-CT) (Figure 3A). Vascularization of a processed bovine cancellous bone (PBCB) matrix was significantly higher in the loop group compared to the group without vascularization. A constantly growing...

Discussion

For over a decade, we have successfully used the arteriovenous (AV) loop for tissue engineering purposes and studying angiogenesis in vivo in the small animal model. We could demonstrate that this microsurgical model is very well suited for engineering different tissues and that it can also be used for angiogenesis or antiangiogenesis studies.

Significance of the Technique with Respect to Existing/Alternative Methods
Engineered tissues or organs require a functi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% sodium chloride	Berlin-Chemie AG	34592508
11-0 Ethilon / polyamide 6/6	Ethicon	EH7438G
4-0 Vicryl / polygalactin 910	Ethicon	V392H
6-0 Prolene / polypropylene	Ethicon	8695H
aluminium spray	Pharma Partner Vertriebs-GmbH	1020
antiseptics	BODE Chemie GmbH
Catheter	B Braun Meslungen AG	4251612-02
contrast agent	Flowtech	MV-122
embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution	Intervet International GmbH
encre de chine intense Indian ink	Lefranc & Bourgeois
Enrofloxacin	Bayer AG
eye ointment	Bayer AG
Formalin 4%	Carl Roth GmbH & Co. KG	P087.4
Heparin	Ratiopharm GmbH
isoflurane	Abbott Laboratories	6055482
Lewis rat, male	Charles River Laboratories
Metamizol-Natrium	Ratiopharm GmbH
papaverine / Paveron N	Linden Arzneimittel-Vertrieb-GmbH
tramadol / Tramal	Grünenthal GmbH