Different Animal Models and Their Importance in Researching Atherosclerosis

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Table of contents

  1. Flow Cytometry
  2. Immunohistochemistry
  3. Conclusion

Atherosclerosis disease is major cause of mortality and morbidity across the world. Many animal models are designed to study atherosclerosis, all experimental conditions, environmental risk factors, diet was optimized. Such animal models are designed with aim of knowing disease’s molecular nature and pathophysiological mechanisms, leading towards provision of platforms for pharmacological development. So, various animal models are developed to answer different sets of questions, for each model various advantages and disadvantages are associated. 1st animal model was rabbit, which was developed to study atherosclerosis disease, leading to identification and investigation of increased level of plaques associated with atherosclerosis. Before development of genetically modified mice, rabbit model served as mainstay of pre- clinical model (Lee et al., 2017). Initially New Zealand White (NZW) strain is developed and its most common. This strain is not prone to atherosclerotic risk because of its less availability of cholesterol level in plasma when exposed to standard diet.

For development of complex atherosclerotic plaques with lipid core, strain must be exposed to cholesterol feeding for longer period i.e., six months to several years. This diet has negative effects, it causes hepatic toxicity increasing mortality (Bocan et al., 1993, Spagnoli et al., 1991). Later, genetically modified rabbits were produced with aim of spontaneous production of atherosclerotic lesions. Watanabe hereditary hypercholesterolemic rabbit (WHHL) is an example of genetically modified rabbit, more prone to coronary atherosclerosis (Shiomi et al., 1992). Both mice and rabbit have small size hence they can be handled easily. They are not only easily available but also have low economical cost. And they share same lipoprotein metabolism with humans. Major disadvantage associated with use of rabbit is that they are not always responsive to cholesterol diet, plaque dissimilarity with humans (Bocan et al., 1993). Wild type and genetically modified porcine models are also developed. Wild type porcine has natural mutation in ApoB and LDLR genes, produced through selective breeding (Civelek et al., 2011). But use of pigs is limited due to its large size, now genetically modified mini pigs are produced but they are not cost effective (Agarwala et al., 2013, Davis et al., 2014).

Non-human primate models show closet similarity to humans as compared to other animal models, showing 98% genetical identity with humans. Despite of closer similarity with humans they are less common due to their larger size, less availability. They are not cost effective and their accommodation require special facilities (Lee et al., 2017).

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Flow Cytometry

Term “flow cytometry” describes measurement of single cell (cyto) as cell flows through multiple detectors, where biophysical properties of each cell can be measured at a rate of thousand cells per second. Flow cytometry technique is used to measure simultaneous measurements of multiple properties of individual cell, while cell flows in suspension through measuring apparatus. Wallace Coulter in 1950s invented 1st original Flow cytometer known as coulter counter. Currently available automated, non-slide-based hematology devices are known to be cytometers. Flow cytometry utilizes beam of laser light that passes through suspension containing cells. when light strikes with cells, it produces signals ultimately detected by detector. Signals are converted to statistical data on computer, where they are presented in different graphical formats (Picot et al., 2012). Basically, flow cytometer operates on principle of using cells in suspension which move through device. So according to this principle blood is ideal tissue having cells already present in suspension and can easily be analyzed using flow cytometry. Flow cytometry uses fluorescence measurements commonly uses laser light. In laser flow cytometers light scattering pattern is used to measure granularity and native size of cell (Bakke, 2001).

Flow cytometry is now highly applicable in medical and biological sciences. Complexity and cost is being decreasing gradually because its analytic ability is increasing (Riley et al., 1993). Most important application of flowcytometry is cellular antigen’s identification and quantification via fluoro chrome-labeled monoclonal antibodies known as immunophenotyping (Alamo and Melnick, 2000, Brown and Wittwer, 2000). Solid organ transplantation including pre- transplantation cross-matching, HLA antibody screening, post transplantation antibody monitoring are done by using flow cytometry analytical technique (Horsburgh et al., 2000, Kupatawintu et al., 2016, Rebibou et al., 2000, Rodrıguez et al., 2000). Flow cytometry is not only used in bone marrow transplantation, in enumeration of CD34+ , to check efficacy of ex vivo T-cell graft depletion, for pre-transplantation but also applicable in post transplantation evaluation of graft rejection, immune recovery, graft-versus host disease and graft versus leukemia disease (Kishino et al., 2002, Lamb, 2002, Storek et al., 2001). Flow cytometry has applications in microbiology, modern flow cytometry allows detection of single and multiple microbes via easy and fast on basic of unique cytometric parameters. Modern Future prospective of this technique would be flow cytometric analysis of apoptosis multi drug analysis, cytokine receptor, leukemia-specific chimeric proteins, revealing new set of information (Álvarez-Barrientos et al., 2000, Shapiro, 2000, Steen, 2000).

In vivo phage display approach is known to be promising bio-panning procedure used to evaluate molecular repertoire of Diseased Endothelial and Subendothelial Tissues present in living animals. In this paper researchers reported application of semisynthetic human single chain Fragment variable library to investigate what is actual molecular mechanism is involved within atherosclerotic lesions under in vivo conditions. This in vivo selection method via its wide panel of pathological markers targeted vasculature, offering a wide opportunity of novel ligand/target pairs (Arap et al., 2002, Krag et al., 2006, Rajotte et al., 1998). In vivo phage display selection is a potent strategy for direct identification of agents targeting vasculature of normal or diseased tissues in living organisms (Deramchia et al., 2012). To identify a panel of individual human scFv-phage having specificity for proteins over represented in atheroma plaques, high through put in vitro screening step against atheroma extracted proteins is necessary.

Screening of selected clones is one of major drawback of in vivo selection method. If antigen is unknown then use of classical ELISA method would not work, it requires purified antigens coated on plates at a high concentration. ELISA is a biochemical assay that uses direct or indirect method to detect antigen it involves adhesion or immobilization of antigen or antigen-specific capture antibody directly on surface of well, respectively (Dobrovolskaia et al., 2006, Gan and Patel, 2013). Alternative to ELISA high through put flow cytometry plaque strategy is proposed via using soluble tissue extracts that is coupled to magnetic bead. A method to screen out and isolation of specific clone from library of phage display flow cytometry is used, this approach allows detection in real time along with isolation of clone having highest affinity for antigen (Feldhaus et al., 2003). This approach is more sensitive as compared to ELISA, needs a little amount of biological material. 200ng of antigen is loaded per well to screen out individual clone, that is 5fold less material is needed comparing with enzyme linked immunosorbent assay. Basically, ELISA method on purified proteins confirms binding specificity. One of major benefit of using high through put flow cytometry is that it allows screening of thousands of clones obtained from bio panning. To carry out efficient screening of scFv-phages using flow cytometry, rate of coupling of protein extracts is important to know.


Immunohistochemistry with selected scFV-phage clones confirms robustness of flow cytometry screening, provide visual localization of these pre-screened clones present in atheroma (Hemadou et al., 2018). For detection of specific antigen immunohistochemistry uses monoclonal and polyclonal antibodies antigen, playing its unique role in different medical fields like in pathology, neuropathology, and hematopathology. Its utilization is rare but now it has been used in surgical pathology (Ajura et al., 2007, Edgar and Rosenblum, 2008, Garcia and Swerdlow, 2009, Leong and Wright, 1987). Immunohistochemistry detects specific antigen using antigen-antibody reaction. Use of IHC has benefit over other traditional techniques which identifies only limited no of proteins, enzymes etc. IHC is being used in drug development to check out drug efficacy (Duraiyan et al., 2012, Rajendran, 2009).


Atherosclerosis plaques are injurious to health. It is necessary to know disease’s molecular nature and pathophysiological mechanisms. To investigate it different in vivo and in vitro studies are conducted. For in vivo phage display for atherosclerosis, different animal models were developed like rabbit, mice, pig etc., each model has its own advantages and disadvantages. General criteria for choosing a suitable animal includes its, size, easy breeding and housing, analogies with humans. ScFv (single chain fragment variable) format consists of variable regions of heavy and light chains, which are joined together by a flexible peptide linker. These human scFv phages targeting atherosclerosis lesions (originally induced in rabbit model) are screened out. Flow cytometry analysis to screen out human scFv phages by in vivo display method using rabbit animal model for atherosclerosis, is an innovative approach. Immunohistochemistry analysis confirms robustness of an innovative flow cytometry analysis. Combining in vivo phage display with flow cytometry analysis, displays its successful application in targeting atherosclerosis antigens in near future.

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