Chick Embryo Partial Ischemia Model: A New Approach to Study Ischemia Ex Vivo Syamantak Majumder, M. Ilayaraja, Himabindu Reddy Seerapu, Swaraj Sinha, Jamila H. Siamwala, Suvro Chatterjee* Vascular Biology Lab, AU-KBC Research Centre, Anna University, Chennai, India Abstract Background: Ischemia is a pathophysiological condition due to blockade in blood supply to a specific tissue thus damaging the physiological activity of the tissue. Different in vivo models are presently available to study ischemia in heart and other tissues. However, no ex vivo ischemia model has been available to date for routine ischemia research and for faster screening of anti-ischemia drugs. In the present study, we took the opportunity to develop an ex vivo model of partial ischemia using the vascular bed of 4th day incubated chick embryo. Methodology/Principal Findings: Ischemia was created in chick embryo by ligating the right vitelline artery using sterile surgical suture. Hypoxia inducible factor- 1 alpha (HIF-1a), creatine phospho kinase-MB and reactive oxygen species in animal tissues and cells were measured to confirm ischemia in chick embryo. Additionally, ranolazine, N-acetyl cysteine and trimetazidine were administered as an anti-ischemic drug to validate the present model. Results from the present study depicted that blocking blood flow elevates HIF-1a, lipid peroxidation, peroxynitrite level in ischemic vessels while ranolazine administration partially attenuates ischemia driven HIF-1a expression. Endothelial cell incubated on ischemic blood vessels elucidated a higher level of HIF-1a expression with time while ranolazine treatment reduced HIF-1a in ischemic cells. Incubation of caprine heart strip on chick embryo ischemia model depicted an elevated creatine phospho kinase-MB activity under ischemic condition while histology of the treated heart sections evoked edema and disruption of myofibril structures. Conclusions/Significance: The present study concluded that chick embryo partial ischemia model can be used as a novel ex vivo model of ischemia. Therefore, the present model can be used parallel with the known in vivo ischemia models in understanding the mechanistic insight of ischemia development and in evaluating the activity of anti-ischemic drug. Citation: Majumder S, Ilayaraja M, Seerapu HR, Sinha S, Siamwala JH, et al. (2010) Chick Embryo Partial Ischemia Model: A New Approach to Study Ischemia Ex Vivo. PLoS ONE 5(5): e10524. doi:10.1371/journal.pone.0010524 Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America Received November 10, 2009; Accepted April 16, 2010; Published May 7, 2010 Copyright: 2010 Majumder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from KB Chandrasekhar- Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: soovro@yahoo.ca Introduction Ischemia is a condition in which blockade in blood flow leads to restricted oxygen and nutrient supply to a part of the body. Cardiac ischemia is the compromised blood flow and oxygen supply to heart muscle. The aim of developing ischemic animal model is to study the basic processes or potential therapeutic interventions in ischemia associated diseases, and the extension of patho-physiological knowledge, which will lead to improve medical treatment of human ischemic stroke. Ischemic stroke has a complex pathophysiology involving the interplay of many different cells and tissues such as neurons, glia, endothelium, cardiomyocytes and the immune system. These events cannot be mimicked satisfactorily in in vitro. Thus a large portion of stroke research is conducted on animals. Presently two ischemic animal models are well accepted by the scientific community. Artery ligation model is created in animal heart by ligating the left coronary artery [1]. Occlusion model is another well accepted model of ischemia, which is created by permanently occluding the middle cerebral artery [2]. However, these different animal models are less feasible for routine laboratory testing of ischemia effects. Additionally, preliminary screening of any anti-ischemic drug needs to be carried out by using in vitro models or by using other models similar to in vivo conditions. In the present scenario, only few in vitro ischemia models such as cerebral ischemia model based on organotypic hippocampal slice cultures and tissue culture ischemia model of oxygen/glucose deprivation are documented [3,4]. However, the models are not well conceived and mostly control the oxygen level to create hypoxia in the tissue but not specifically ischemia. In this present study, we carried out experiments to develop a novel ex vivo partial ischemia model by blocking the right vitelline arteries of chick embryo. The model was validated by using three tier experimental set ups 1) ischemia in the vascular bed 2) secondary ischemia in endothelial cells (EC) and cardiomyocyte cells cultured on ischemic vascular bed and 3) secondary ischemia created in caprine cardiac tissues by placing them on ischemic vascular bed. Elevated level of reactive oxygen species (ROS) and HIF-1a followed by deteriorated cell and tissue functions validated the chick embryo partial ischemia model. Further, a strong PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10524