Figure 8. RL based recovery of GSH level in ischemic vitelline vascular tissue. (A) Level of free thiol in ischemic tissues treated with and with out RL were measured using CMFDA. Ischemic vessels were incubated with 100 mM of RL during the period of treatment. Next, the ischemic tissues were incubated with CMFDA for 30 min and imaged with DP71 CCD camera. Images are the representative of 5 individual experiments. (B) The fluorescent intensities of the images were calculated using Adobe Photoshop 7.0 and plotted. Fluorescent intensities were considered as directly proportional to the level of free thiol in tissues. **P,0.001 versus non-ischemic and yyP,0.001 versus ischemia. (n = 5). doi:10.1371/journal.pone.0010524.g008 tissue on the ischemic vascular bed. To confirm ischemia in the heart tissues, we measured the activity of CK-MB, a known marker of myocardial ischemia. Biochemical analysis of the heart tissue sample depicted that ischemic vascular bed mediated ischemia in caprine heart tissues elevated the enzyme activity of CK-MB by 72% (Fig. 14A). Elevation of CK-MB activity in caprine heart elucidated that ischemic vascular bed of chick embryo successfully induced secondary ischemia in caprine heart tissues. Partial ischemia caused edema and higher intra-muscular gap in caprine heart Caprine heart tissues were incubated on the ischemic vascular bed and fixed with 10% formalin. Parafilm wax embedded tissue sections were stained using hematoxylin and eosin stains. Images of the sections depicted that ischemic vascular bed promoted edema and an elevated intra-mascular gaps in caprine heart tissue (Fig. 14B). This data suggested that chick embryo ischemic vascular bed induced secondary ischemia in caprine heart tissue. Discussion Different animal models are available for tissue specific ischemia such as embolic middle cerebral artery occlusion and endovascular Chick Embryo Ischemia Model filament middle cerebral artery occlusion. Middle cerebral artery occlusion is achieved by injecting particles like blood clots or artificial spheres into the carotid artery of animals as an animal model of ischemic stroke. Thromboembolic MCAo is achieved either by injecting clots that were formed in vitro [6] or by endovascular instillation of thrombin [7]. The thromboembolic model is closest to the pathophysiology of human cardioembolic stroke. But, the fundamental problem in ischemia research is the lack of easy to develop routine research models, which should offer a regular testing platform for routine research and drug testing for ischemia. The present study focused to develop a partial ischemia model by using vascular bed of 96 h grown chick embryo. The present work aimed to develop a basic ischemia model instead tissue or organ specific one, which would cater the scientists in academic and industry to understand the fundamental aspects of ischemia implications and will also help in faster screening of anti- ischemic drugs. Although chick embryo never been used before for creating ischemia, efforts were made to develop chick embryo based flow-manipulation model for shear stress associated research [8–10]. Hogers et al., 1999 have taken two approaches to manipulate the blood flow in chick embryos [8]. First, one of the three major vitelline veins was ligated and secondly these vitelline veins were ligated permanently with a microclip until cardiac septation was completed. This venous clip model is one of the major approaches to control the blood flow in the vascular bed of chick embryo [8,9]. However, these studies are directed towards evaluating the consequences of low blood flow on embryo development and cardiovascular malformations while the present study aims to develop an ex vivo partial ischemia model by using a similar approach to control the blood flow in chick embryo vascular bed. We ligated the right vitelline artery of chick embryo that blocks the blood-flow of corresponding areas of vascular bed in the chick embryo. Work of Lee et al., 2000 and different other groups suggested that an early stage elevation of HIF-1a expression under myocardial ischemia is a protective measure by the cell to protect itself from the ischemia induced damage [11–16]. We measured the level of HIF-1a expression in the ligated chick embryo model by considering it as one of the early markers of ischemic tissue damage, and we also found that HIF-1a expression level elevated in affected vascular bed and also in cells cultured on the ischemic vascular bed. The work of Yamamoto et al (1983) demonstrated that lipid peroxidation plays a major role in ischemia driven cellular damages [17]. Works of different groups have also reported that a higher level of lipid peroxidation during ischemia and ischemic reperfusion causes cellular damage and cell death [17–19]. Yamamoto et al., 1983 have shown that administration of a-tocopherol, a ROS scavenger and a blocker of lipid peroxidation improves the cellular health under ischemia [17]. Similarly EC incubated on the ischemic vascular bed of chick embryo evoked a higher level of lipid peroxidation indicating that primary ischemia in the vascular bed spreads secondary ischemia in the cells and tissues grown on the affected vascular bed. ROS generation during ischemia is a consequence of ischemia mediated tissue damage [20–22]. The burst of ROS during ischemia reperfusion is considered as one of the key factors in the development of myocardial stunning after regional ischemia [23,24]. Generation of ROS during ischemia [25–27] are considered as an important factor in the development of cardiac preconditioning [28,29]. Work of Becker et al., 1999 concluded that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain [25]. All together these studies signify that ischemia induced ROS generation by cellular systems plays a major role in ischemia mediated tissue PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10524