Animal Models Used to Study Atherosclerosis

Different animal models and their importance:

Atherosclerosis disease is a major cause of mortality and morbidity across the world. Many animal models are designed to study atherosclerosis, all experimental conditions, environmental risk factors, and diet was optimized. Such animal models are designed with aim of knowing the disease’s molecular nature and pathophysiological mechanisms, leading toward the 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 a rabbit, which was developed to study atherosclerosis disease, leading to the identification and investigation of increased levels of plaques associated with atherosclerosis. Before the development of genetically modified mice, the rabbit model served as the mainstay of the pre-clinical model. 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 levels in plasma when exposed to a standard diet. For the development of complex atherosclerotic plaques with a lipid core, strain must be exposed to cholesterol feeding for a longer period i.e., six months to several years. This diet has negative effects, it causes hepatic toxicity increasing mortality. Later, genetically modified rabbits were produced with aim of spontaneous production of atherosclerotic lesions. Watanabe's hereditary hypercholesterolemic rabbit (WHHL) is an example of a genetically modified rabbit, more prone to coronary atherosclerosis. Both mice and rabbits have small sizes hence they can be handled easily. They are not only easily available but also have low economical cost. And they share the same lipoprotein metabolism with humans. The major disadvantage associated with the use of rabbits is that they are not always responsive to a cholesterol diet, and plaque dissimilarity with humans. Wild type and genetically modified porcine models are also developed. Wild-type porcine has a natural mutation in ApoB and LDLR genes, produced through selective breeding. But the use of pigs is limited due to its large size, now genetically modified mini pigs are produced but they are not cost-effective.

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

Flow Cytometry

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

Flow cytometry is now highly applicable in medical and biological sciences. Complexity and cost is being decreasing gradually because its analytic ability is increasing. The most important application of flow cytometry is cellular antigen identification and quantification via fluorochrome-labeled monoclonal antibodies known as immunophenotyping. Solid-organ transplantation including pre- transplantation cross-matching, HLA antibody screening, and post-transplantation antibody monitoring is done by using the flow cytometry analytical technique. Flow cytometry is not only used in bone marrow transplantation, in the enumeration of CD34+, to check the efficacy of ex vivo T-cell graft depletion, for pre-transplantation but is also applicable in the post-transplantation evaluation of graft rejection, immune recovery, graft-versus-host disease and graft versus leukemia disease. Flow cytometry has applications in microbiology, modern flow cytometry allows the detection of single and multiple microbes via easy and fast on basic of unique cytometric parameters. Modern Future prospective of this technique would flow cytometric analysis of apoptosis multi drug analysis, cytokine receptor, and leukemia-specific chimeric proteins, revealing a new set of information.

In vivo phage display approach is known to be a promising bio-panning procedure used to evaluate the molecular repertoire of Diseased Endothelial and Subendothelial Tissues present in living animals. In this paper, researchers reported the application of a semisynthetic human single-chain Fragment variable library to investigate what 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. In vivo phage display selection is a potent strategy for the direct identification of agents targeting the vasculature of normal or diseased tissues in living organisms. To identify a panel of individual human scFv-phage having specificity for proteins over-represented in atheroma plaques, high throughput in vitro screening step against atheroma extracted proteins is necessary. Screening of selected clones is one of the major drawbacks of in vivo selection method. If the antigen is unknown then the use of the 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 methods to detect antigens it involves adhesion or immobilization of antigen or antigen-specific capture antibody directly on the surface of a well, respectively. Alternative to ELISA high throughput flow cytometry plaque strategy is proposed via using soluble tissue extracts that are coupled to a magnetic bead. A method to screen out and isolation of specific clones from the library of phage display flow cytometry is used, this approach allows detection in real-time along with isolation of clones having the highest affinity for antigen. This approach is more sensitive as compared to ELISA and needs a little amount of biological material. 200ng of antigen is loaded per well to screen out individual clones, that is 5fold less material is needed compared with enzyme-linked immunosorbent assay. Basically, the ELISA method on purified proteins confirms binding specificity. One of the major benefits of using high throughput flow cytometry is that it allows the screening of thousands of clones obtained from biopanning. To carry out efficient screening of scFv-phages using flow cytometry, the rate of coupling of protein extracts is important to know.

Immunohistochemistry

Immunohistochemistry with selected scFV-phage clones confirms the robustness of flow cytometry screening and provides visual localization of these pre-screened clones present in atheroma. For detection of specific antigens, 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. Immunohistochemistry detects specific antigens using antigen-antibody reactions. The use of IHC has a benefit over other traditional techniques which identifies only a limited no of proteins, enzymes, etc. IHC is being used in drug development to check out drug efficacy.

Conclusion 

Atherosclerosis plaques are injurious to health. It is necessary to know the 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 rabbits, mice, pig,s etc., each model has its own advantages and disadvantages. General criteria for choosing a suitable animal include its, size, easy breeding and housing, and 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 the 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 the 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.