recovery of ischemia by anti-ischemic drugs validated our novel approach of developing an ex vivo ischemia model. Results Partial ischemia elevated the reactive oxygen species generation in tissues Different ROS parameters like superoxide, hydrogen peroxide, peroxynitrite and lipid peroxidation. were evaluated in the ischemic vessels of chick embryo (Fig. 1) using different redox probes. Mapping of lipid peroxidation was carried out using the thiobarbituric acid (TBA) protocol. A higher level of lipid peroxidation was observed in the proximal areas in relation to the ischemic zone while lipid peroxidation level reduced in distal areas (Fig. 2A,2B). Image analysis and colorimetric measurement of superoxide in ischemic tissues demonstrated 40% and 42% increase in superoxide level respectively (Fig. 3). Measurement of hydrogen peroxide using amplex red depicted that ischemia attenuated hydrogen peroxide production by 25% (image analysis) and 27% (fluorimetric measurement) respectively (Fig. 4). Nitric oxide (NO) level in the ischemic vessels was measured using diamino fluorescein diacetate (DAF-2DA) fluorescence probe. There was no significant difference in NO production between ischemic and non-ischemic vessels was observed (Fig. 5A,5B). Peroxynitrite generation in the ischemic vessels was measured using dihydrorhodamine (DHR123) fluorescence probe. Partial ischemia elevated the peroxynitrite level in tissue by 33% compared to non-ischemic controls (Fig. 5C). Image analysis and Chick Embryo Ischemia Model fluorimetric measurement of free thiol in ischemic tissues using 5- chloromethylfluorescein diacetate (CMFDA) elaborated 48% and 42% reductions respectively in free thiol level under partial ischemia treatments (Fig. 6). Partial ischemia dependent higher HIF-1a release induced the neo-vascularization in the adjacent vessels HIF-1a, a hypoxia driven factor, is a well known pro-angiogenic factor as reported earlier [5]. We evaluated the concept and studied the angiogenesis pattern of the vascular bed adjacent to the ischemic area. Analysis of the vascular bed images using Angioquant software revealed that ischemia elevated the neo- vascularization in chick embryo vascular bed in a time dependent manner. Analysis of number of vessel complexes, vessel length, vessel size and junctions revealed 4, 5.5, 4.5 and 11 fold increase respectively after the ischemia treatment (Fig. 7). Increase in vascularization can also be due to backfilling of initially occluded vessels. However, critical analysis of the images from ischemic vessels showed that the vessels were formed newly from the existing vessels and thus can be concluded that increase in the vessel density is due to the neo-angiogenesis in that area. Partial ischemia in chick embryo vessels elevated the expression level of HIF-1a in a time-dependent manner HIF-1a expression was used as a biochemical marker to quantify the level of ischemic stress in tissues. Relative expression of HIF-1a in ischemic vessels was measured using quantitative Figure 1. Creating partial ischemia in chick embryo vascular bed using ligation model. (A) Image showed the embryo with the whole yolk material has been placed in the sterile glass bowls. The bowls were covered with sterile glass leads and placed in a chamber maintaining 37uC and relative humidity. (B) Image represents the exact approach taken to block the blood flow to the specific area of the chick embryo vascular bed. Black arrow showed the suture that has ligated the right vitelline artery thus blocking the blood flow in the surrounding vascular bed. The box showed the ischemic area of the vascular bed. doi:10.1371/journal.pone.0010524.g001 PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10524