**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.


*Edited by George Dekoulis*

This edited volume "Field-Programmable Gate Array" is a collection of reviewed and relevant research chapters, offering a comprehensive overview of recent developments in the field of semiconductors. The book comprises single chapters authored by various researchers and edited by an expert active in the aerospace engineering systems research area. All chapters are complete within themselves but united under a common research study topic. This publication aims at providing a thorough overview of the latest research efforts by international authors and open new possible research paths for further novel developments.

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Field - Programmable Gate Array

Field

Programmable Gate Array

*Edited by George Dekoulis*


**Author details**

Khagani Mammadov Institute of Radiation Problems of Azerbaijan National Academy of Sciences, Baku, Azerbaijan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Study of Sources of Drinking Water and Processes of Water's Purification from Pollutants DOI: http://dx.doi.org/10.5772/intechopen.113735*

### **References**

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[3] Neta P, Grodkowski J. Rate constants for reactions of phenoxyl radicals in solution. The Journal of Physical Chemistry. 2005;**34**(1): 109-199

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[11] Mammadov K. Effect of natural radionuclides on the processes occur in the environmental objects. Studying the cleaning methods of water contaminated with organic emissions and the methods for cleaning of soil contaminated with heavy metals and radionuclides. International Journal of Applied Chemistry. 2020;**7**(01):18-24. DOI: 10.14445/23939133/IJAC-V7I1P105

[12] Piskunov AS. Methods of Agrochemical Research. Moscow: Kolos; 2004. 312 p

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[14] Mamedov KF. Research of Distribution of Heavy Metals and Radioactive Elements in Sources of Drinking Water, Soil and Grass Cover of Azerbaijan Regions. Petrozavodsk: Tsifra; 2020. 43 p. ISBN: 978-5- 901619-31-5

[15] Mamedov KF. Radiolysis and photolysis of water solutions of phenol. European Researcher. Series A. 2014;**7-1**: 1216-1236. DOI: 10.13187/issn.2219-8229

[16] Mammadov KF. Determining the sources of contamination of the waters discharged into the Araz river with inorganic xenobiotics and pathogenic microorganisms. Pedagogy University News. Mathematics and Natural Sciences Series. 2020;**3**:168-185. ISSN: 2520-2049

[17] Mamedov KF. Radiolytic conversion of natural toxins in contaminated plant products and aqueous solutions.

Scientific Journal "Science Rise". 2014; **4**(2):116-121. DOI: 10.15587/ 2313-8416.2014.29402

[18] Mammadov XF. Kinetics of formation of nitrites in chlorinated waters of closed water sources. Scientific Works – Fundamental Sciences. Chemistry. 2012;**1**(41):163-165. ISSN: 1815-1779

[19] Mahmudov HM, Kuliyeva UA, Kerimov VK, Kurbanov MA. Water radiolysis on the surface of Al2O3 nanocatalyst. European Journal of Analytical and Applied Chemistry. 2015;**1**:58-62. DOI: 10.1155/2021/9493765

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[22] Sakumoto A, Miyata T. Treatment of waste water by a combined technique of radiation and conventional method. Radiation Physics and Chemistry. 1984; **24-1**:99-115


**4. G-quad as fluorescence probes**

The photophysical properties of G-quad structures are highly dependent on the bound metal ions at the location of nucleobase electrons of the structure due to the coordination of ions in the center of the quadruplex. Thus, G-quad is able to exhibit two- to ten-fold higher quantum fluorescence yields. This is due to the structure serving as energy donors to energy acceptors in close proximity to yield fluorescence resonance energy transfer (FRET) systems. G-quad can be utilized as a fluorescent probe in which the nucleobases in the structure are labelled or attached with fluorescent dyes. Besides, the enhancement of the fluorescence of a small dye can be exhibited based on the interaction of the G-quad with the fluorescence ligand [7,37,38]. Some of the ligands such as thiazole orange were reported to yield higher fluorescence yield with the aid of the G-quad structure [39]. On the other hand, the G-quad could also cause fluorescence quenching phenomena. For instance, the classic intercalating agent, ethidium bromide (EtBr), binds to double-stranded DNA and exhibit fluorescence intensity up to 30 fold. However, in the presence of G-quad, the fluorescence intensity of the EtBr can be quenched upon binding to the structure [7].

generated hemin–DNAzyme complex will catalyse the oxidation of ABTS to produce a coloured product [35]. Isothermal DNA amplification strategies can be employed easily for

Besides RCA, G-quad structures can also be amplified using other isothermal strategies like quadruplex priming amplification (QPA) as reported by Kankia et al. QPA allows for efficient generation of G-quad structures by isothermal amplification with the additional ability to selfdissociate for continuous amplification. During the QPA elongation process, the 5' end of the product will fold into intramolecular quadruplex and self-dissociates from the primer-binding site of the template, allowing the template to be accessible to the incoming primer for the next priming cycle. Thus, more quadruplexes were formed and eventually leading to increased signal readout. Our group incorporated the QPA system for the development of a sandwich immunoassay, called immuno-QPA (IQPA). The system exploits the peroxidase mimicking function of G-quad DNAzyme and the quadruplex amplification by QPA. A recombinant scFv was coated on the well and the biotinylated antigen was added and left to bind with the antibody. Streptavidin was introduced as a bridge between the biotinylated antigen and biotinylated QPA template. QPA was then carried out to amplify the QPA template with its specific primer to generate self-dissociating G-quad structures. These self-dissociating G-quad structures will bind to hemin to generate the similar colour change readout with ABTS as

The photophysical properties of G-quad structures are highly dependent on the bound metal ions at the location of nucleobase electrons of the structure due to the coordination of ions in

DNAzyme sensor detection.

126 Nucleic Acids - From Basic Aspects to Laboratory Tools

mentioned earlier [36].

**4. G-quad as fluorescence probes**

**Figure 3.** Ultrasensitive detection of thrombin by RCA and G-quad DNAzyme.

One of the label-free G-quad DNA-based fluorescence biosensors reported was designed to detect cisplatin, which is an anticancer drug widely used in chemotherapy. However, cisplatin overdose can cause neurotic cell death that makes it dangerous for administration without proper monitoring. Therefore, Zhou et al. developed a simple label-free fluorescence biosensor by utilizing the interaction of G-quad and cisplatin as cisplatin was found to bind to G-quad naturally (Figure 4). In the context of the assay, the absence of cisplatin will allow the formed G-quad to bind to N-methyl mesoporphyrin IX (NMM), resulting in an increase in fluorescence intensity. However, when cisplatin was added into the assay, it will bind to the G-quad structure and disintegrate the structure. This will then cause a drastic decrease in fluorescence intensity due to the collapse of the G-quad structure [40].

**Figure 4.** Label-free turn on and turn off fluorescence DNA G-quad-based sensor for the detection of cisplatin.

Wang et al. had proposed a label-free fluorescence biosensor based on G-quad formation in order to detect the lead (II) ion (Pb2+). This biosensor consists of a G-rich DNA strand with its partially complementary strand. The heme-oxygenase-1 inhibitor, zinc protoporphyrin (ZnPPIX) has been utilized as a fluorescence probe, whereby it can interact with the Pb2+/G– quad complex, producing fluorescence readout. In the absence of the Pb2+ ions, both DNA strands will form a DNA duplex. The addition of Pb2+ will unwind the DNA duplex to allow the G-rich strand to fold into the G-quad structure. The Pb2+/G–quad complex will then interact with ZnPPIX to enhance its fluorescence intensity, which is not possible with the DNA duplex. This biosensor could overcome the cumbersome step in which most of the fluorescence-based biosensors would require fluorescence dyes to be probed on the DNA. In addition, this biosensor can be reset easily to the original state by dissociating the G-quad structure. In order to dissociate the G-quad structure, the strong Pb2+ chelator DOTA (1,4,7,10-tetraazacyclodo‐ decane-1,4,7,10-tetraacetic acid) was used as it has high binding capacity against Pb2+ to remove Pb2+ from the G-quad structure, leaving the G-quad structure to dissociate and form a DNA duplex with the complementary strand again [41].

As reported by Kankia and co-workers, a further improvement to their original QPA method was reported [42]. The improved method involves the use of two linear processes that are the QPA and linear nicking amplification, which is based on the study done by Galas and coworkers. The probe DNA was hybridized with the target DNA and polymerase was used to extend the target DNA strand and form QPA-PBS (primer-binding site). Then, the nicking enzyme Nt.BSTNBI was introduced to nick the target strand and release the QPA-primerbinding strand. The QPA-primer-binding strand will bind to the QPA primer and the poly‐ merase will function to extend the strand further. The primer will then dissociate to emit fluorescence signals. The following priming step of QPA will be initiated again when the next primer binds with the QPA-primer binding strand. This method was reported to be able to reduce the background activity to allow sufficient sensitivity of the assay (Figure 5).

**Figure 5.** Principle of the QPA integrating nicking enzyme for diagnostics.