**5. Rat ischemic hind limb model**

744 Biomedical Science, Engineering and Technology

the development of therapeutic agents in the prevention and treatment of cancer

Among the list of lymphangiogenic factors, members of the VEGF family are the best characterized. VEGF-A, which binds to VEGFR-2 and VEGFR-1, PlGF and VEGF-B which bind to VEGFR-1 and especially VEGF-C and VEGF-D which bind to VEGFR-3 are lymphangiogenic factors, the -C and -D isoforms being the most potent, which regulate both physiological and pathological lymphangiogenesis. VEGF-C/VEGF-D-VEGFR-3-mediated signals are also critical for the sprouting of the first lymphatic vessel from the developing veins in the embryo. This signaling pathway is essential for differentiation of endothelial progenitor

Primary tumors produce several lymphangiogenic factors, including VEGF-A, VEGF-C, VEGF-D, Insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and PDGF-B. These factors induce angiogenesis and lymphangiogenesis both in the local environment and in the regional lymph nodes. Furthermore, they play an important role in establishing the pre-metastatic niche. The term pre-metastatic niche describes the adaptations of for example the lymph nodes which are needed to allow disseminating tumor cells, arriving at a later stage, to meet optimal conditions for growth in that particular site. In addition to signals produced by the tumor cells themselves, inflammatory cells such as macrophages are recruited to tumors by a wide range of tumor cell-derived cytokines and growth factors. At the tumor site, inflammatory cells play a critical role in mediating lymphangiogenesis

To detect lymphatic vessels in or around tumors, immunohistochemical staining with lymphatic specific markers, such as VEGFR-3, LYVE-1 and podoplanin is recommended (see Fig. 13). Lymphangiogenesis can be evaluated using all of the models described in this chapter, by using antibodies against one or a combination of these factors either in whole

As tumor cells often produce a multitude of factors which may cooperate in inducing angiogenesis and lymphangiogenesis, these models are considered to be relatively "dirty" and unsuitable for studying the effects of just one or a few factors. In order to overcome this problem, a xenograft model for non-tumor angiogenesis has been developed. In this model, a matrigel plug is grafted onto the mouse which can be mixed with recombinant angiogenic

Matrigel consist of purified basement membrane components (collagens, proteoglycans and laminin) and, while it is liquid at temperatures just above 0 degrees, it forms a gel when it is warmed to 37 °C. Thus, the material can be cooled and then injected in the mice, where it will form a three dimensional gel, in which host blood vessels can invade. Matrigel itself is a poor inducer of angiogenesis, but it can be mixed with angiogenic growth factors and/or cells prior to injection leading to a controllable induction of blood vessel growth into the plug. Plug vessels are usually evaluated 7-21 days after implantation by gross examination/photography as well as immunohistochemical staining as described above (Akhtar et al, 2002). If the plug contains functional vessels, the blood (red) vessels can be identified from the photograph. Alternatively, by using mice which express GFP in the

cells into the lymphatic lineage (Kukk et al, 1996; Alitalo et al, 2005).

most likely through the secretion of several lymphangiogenic cytokines.

**4. Non-tumor, xenograft models** 

factors prior to grafting.

**4.1 Matrigel plug assay** 

mount staining or on thin sections of frozen or paraffin embedded tissue (Fig. 13).

metastasis.

Most of the models mentioned above are very useful to study pathological angiogenesis, and therefore used to find novel anti-angiogenic treatment options including novel antiangiogenic drugs, but other assays are needed to study diseases where therapy would consist of accelerated blood vessel growth, such as in the treatment of myocardial infarction (MI), stroke and wound healing/regeneration.

In MI an occluded coronary artery leads to a blockade of blood flow to a part of the cardiac muscle tissue, and thus leads to severe hypoxia (ischemia). The cardiac musculature is working constantly, has a very high metabolism and is therefore particularly sensitive to reduced oxygen (and sugar) levels. Therefore, unless new, so called collateral, arteries can be formed quickly, the affected cardiac tissue will perish, and usually so will the patient (Y Cao, 2010). Effective therapy would therefore be able to induce growth of highly functional arteries in the response to tissue hypoxia/ischemia.

An excellent model to study and manipulate the growth of blood vessels and in particular arteries in response to tissue hypoxia is the hind limb ischemia model, which can be performed in either mice or rats. In this model, all arteries supplying highly oxygenated blood to one of the back limbs of the animal are ligated in two steps, resulting in near-zero blood flow in the entire limb (Lundberg et al, 2002.) (Fig. 11). This leads to tissue

Fig. 11. Hind limb ischemia model. First, a midline incision is made (top dashed line) exposing the vessels. All branches originating from the aorta distal to the renal arteries and all branches from the left iliac artery are ligated by a small suture (blue knots). After a week a second inguinal incision is made (bottom dashed line) and the femoral artery and superficial gastric artery are ligated by a small suture (blue knots). Following this operation the left hind limb does not receive any blood and is considered ischemic.

ischemia and the induction of arteriogenesis from collateral arteries. Angiogenic factors under investigation can be injected into the limb musculature or antibodies against angiogenic or anti-angiogenic factors can be injected into circulation, thus modulating the arteriogenic response. As the hind limb is easily accessible, the circulation in the limb can be observed by Doppler-angiography and thus the same animal can be subjected to repeated investigations on how the blood flow improves over time. After euthanasia, the tissue can be excised and stained as mentioned for the adipose tissue, and the morphology of the blood vessels can therefore be studied post mortem (R Cao et al, 2003). This assay is the most commonly used assay to study therapeutic angiogenesis, and has been used in many seminal discoveries on how to therapeutically generate highly functional and stable arteries (Y Cao, 2010). However, the surgery needed to induce ischemia in the hind limb is very complicated and the assay therefore requires highly skilled and experienced surgeons. Also, there may be slight inter-individual differences in the residual blood flow in the limb after surgery – and therefore the degree of tissue hypoxia - within each experimental group.

## **6. Wound healing assay**

The process of wound healing is divided into three stages; inflammation, new tissue formation and remodeling. Angiogenesis is critical for the formation of new tissue and vascular remodeling occurs in association with tissue remodeling at later stages. Therefore, the wound healing assay is a useful model to evaluate both angiogenesis and vascular maturation/remodelling. Wound healing models are usually performed on the skin as other accessible tissues such as the ears or the tail do not regenerate well. Usually, two circular, trans-dermal wounds are created on the back of anesthetized mice (Fig. 12) allowing one wound to serve as control while topical treatment can be administered on the other. Wound size, scar formation and re-epithelization of the wounds should be recorded daily by photography and by measuring the wound area with calipers, similar to how it is done in the tumor models. In this model, treatment given either systemically by oral administration or injection, or preferably topically on just one of the two wounds, can consist of pro- or anti-angiogenic compounds, and their effects on both the regenerative angiogenesis as well as on vessel morphology and function can be determined post mortem after the regenerated tissue has been excised, fixed and stained as mentioned for the adipose tissue. Also, transgenic or knock-out mice can be used, when available, to study the specific effects of particular genes (Xue et al, 2008).

The surgery required to create the wounds is very simple and the wounds are highly homogeneous in size and location for all animals used in the experiments. This assay is therefore very easy and robust, requires little practice and few animals as the experimental variation is relatively low. However, angiogenesis in this model occurs only in the skin, and in association with inflammation, blood clotting cascades and other highly complex biological processes, which encompass multiple cell types and a plethora of angiogenic and anti-angiogenic factors. Furthermore, regeneration of the skin is known to be quite different from regeneration of other tissues of higher clinical relevance such as the heart and nervous system, and can therefore not be expected to give much information on the role of angiogenesis in regeneration of other organs. Another drawback of this assay is that regeneration occurs by making new tissue rather than repairing/replacing damaged/dead tissue. This difference is thought to be important, as the latter is almost always the case in

ischemia and the induction of arteriogenesis from collateral arteries. Angiogenic factors under investigation can be injected into the limb musculature or antibodies against angiogenic or anti-angiogenic factors can be injected into circulation, thus modulating the arteriogenic response. As the hind limb is easily accessible, the circulation in the limb can be observed by Doppler-angiography and thus the same animal can be subjected to repeated investigations on how the blood flow improves over time. After euthanasia, the tissue can be excised and stained as mentioned for the adipose tissue, and the morphology of the blood vessels can therefore be studied post mortem (R Cao et al, 2003). This assay is the most commonly used assay to study therapeutic angiogenesis, and has been used in many seminal discoveries on how to therapeutically generate highly functional and stable arteries (Y Cao, 2010). However, the surgery needed to induce ischemia in the hind limb is very complicated and the assay therefore requires highly skilled and experienced surgeons. Also, there may be slight inter-individual differences in the residual blood flow in the limb after surgery – and therefore the degree of tissue hypoxia - within each

The process of wound healing is divided into three stages; inflammation, new tissue formation and remodeling. Angiogenesis is critical for the formation of new tissue and vascular remodeling occurs in association with tissue remodeling at later stages. Therefore, the wound healing assay is a useful model to evaluate both angiogenesis and vascular maturation/remodelling. Wound healing models are usually performed on the skin as other accessible tissues such as the ears or the tail do not regenerate well. Usually, two circular, trans-dermal wounds are created on the back of anesthetized mice (Fig. 12) allowing one wound to serve as control while topical treatment can be administered on the other. Wound size, scar formation and re-epithelization of the wounds should be recorded daily by photography and by measuring the wound area with calipers, similar to how it is done in the tumor models. In this model, treatment given either systemically by oral administration or injection, or preferably topically on just one of the two wounds, can consist of pro- or anti-angiogenic compounds, and their effects on both the regenerative angiogenesis as well as on vessel morphology and function can be determined post mortem after the regenerated tissue has been excised, fixed and stained as mentioned for the adipose tissue. Also, transgenic or knock-out mice can be used, when available, to study the specific effects of

The surgery required to create the wounds is very simple and the wounds are highly homogeneous in size and location for all animals used in the experiments. This assay is therefore very easy and robust, requires little practice and few animals as the experimental variation is relatively low. However, angiogenesis in this model occurs only in the skin, and in association with inflammation, blood clotting cascades and other highly complex biological processes, which encompass multiple cell types and a plethora of angiogenic and anti-angiogenic factors. Furthermore, regeneration of the skin is known to be quite different from regeneration of other tissues of higher clinical relevance such as the heart and nervous system, and can therefore not be expected to give much information on the role of angiogenesis in regeneration of other organs. Another drawback of this assay is that regeneration occurs by making new tissue rather than repairing/replacing damaged/dead tissue. This difference is thought to be important, as the latter is almost always the case in

experimental group.

**6. Wound healing assay** 

particular genes (Xue et al, 2008).

clinical situations where for example ischemic insults result in large patches of dead tissue that is hard to replace.

Fig. 12. Wound healing model. Two circular holes of approximately 5 mm in diameter are punched with a tissue puncher through the dorsal skin of an anesthetized wildtype C57Bl6 mouse. No bandages or cover is needed, as the skin in this region has no major blood vessels, and the wound formation leads to very little if any bleeding. Wounds close within 2 weeks and would heal completely within a month.
