Chick Embryo Ischemia Model Figure 10. Effect of ischemic vascular bed driven secondary ischemia on EC. (A) The number of round and normal cells were counted from the bright field images. Data were normalized by counting the total number of cells per field and presenting it as percentage of cell. **P,0.001 versus non-ischemic. (n = 7) (B) Number of normal cells were counted from each field and plotted as percentage of normal cells. Ischemia induced reduction in normal cell number was calculated while reversibility was observed when the cells kept back on normal vessels. **P,0.001 versus preischemic and yyP,0.001 versus postischemic. (n = 7). (C) Apoptosis was measured in ischemia treated EC using annexin-V FITC detection kit. The total number of cells and the number green positive cells per field were counted and plotted. Images are the representative of 3 individual experiments. **P,0.001 versus non-ischemic. (n = 3) (D) Cellular viability under ischemia treatment was measured using trypan blue dye. Trypan blue stained cells were taken as dead cells. The total number of cells and the number green positive cells per field were counted and plotted. Images are the representative of 7 individual experiments. **P,0.001 versus non-ischemic. (n = 7). doi:10.1371/journal.pone.0010524.g010 CK-MB activity has been shown to increase in certain patients with ischemic stroke, sub-arachnoid hemorrhage and head trauma in the absence of any clinically evident acute coronary syndrome [31,44–47]. The temporal pattern of elevation was typically gradual and sustained for several days, unlike myocardial infarction, in which CK-MB peaks and falls within the first 24 h of coronary artery occlusion [48]. Similarly, our study depicted that CK-MB activity is high when the caprine heart tissue sample was incubated on the ischemic vascular bed of chick embryo. As reported earlier by Zhai et al., 2000, we also observed a similar histology pattern of the ischemic heart tissue sample where marked interstitial edema and evidence of intramascular gaps were observed when incubated under ischemic vascular bed driven ischemia [49]. These observations demonstrate that ligation mediated partial ischemia in chick vascular bed could create secondary ischemia in the caprine heart tissues as well. The ligation of right vitelline artery in chick embryo causes partial ischemia in the surrounding vascular bed, which can further be used to create secondary ischemia in cells and other tissues under in vitro condition. The present model offers some unique features for routine ischemia related drug testing such as 1) less ethical issues 2) easy to develop model 3) real time observation of physiological parameters such as blood flow and angiogenesis and 4) culturing of cells and tissues of one’s choice on the ischemic bed makes the model very plastic. However, the present model also has limitations in its present form. The limitations are 1) First of all, the model cannot be tissue or organ specific as it is possible in animal models. 2) Secondly, the model cannot produce intermittent ischemia or ischemic reperfusion injury, which is prevalent in human. 3) Unlike adult tissues, the avian embryo during early stages of development obtains oxygen through direct diffusion into the tissues (until day 5–6). In subsequent stages the oxygen diffuses into blood circulating through the extra-embryonic chorioallantoic membrane. However, due to blockade in artery, blood flow to that specific area is blocked which will partially hinder with the diffusion properties of free oxygen to the vessels. Therefore, a partial blockade in both oxygen and nutrient diffusion was obtained in the present model and thus termed as partial ischemia model. Particularly, during dynamic exercise, normal coronary arteries dilate, whereas stenotic arteries constrict. This exercise-induced vasoconstriction has been associated with the occurrence of myocardial ischemia and has been believed to be the result of endothelial dysfunction, with a reduced release or production of NO, increased sympathetic stimulation, enhanced platelet aggregation with release of thromboxane A2 and serotonin or a passive collapse of the disease-free wall segment within the stenosis (the Bernoulli effect) or a combination of any of these [50]. It is not possible to exert exercise like effects on the present model of ischemia. However, work is in progress in our laboratory to modulate the blood flow in a PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10524