Figure 2. Mapping lipid peroxidation in ischemic vascular bed. (A) Representative image showed the way section has been incised out from the vascular bed of chick embryo to map the lipid peroxidation status in the ischemic vascular bed. (B) Lipid peroxidation was measured by following the TBA protocol. After ligation of right vitelline artery, embryos were incubated for 2 h. An equal six sections of the chick embryo vascular bed was taken and the lipid peroxidation was measured. Data were normalized by taking the weight of the tissues.*P,0.05 and **P,0.001 versus C,D,E and F. (n = 5). doi:10.1371/journal.pone.0010524.g002 RT-PCR technique. The vessels were ligated and incubated for different durations ranging from 0 to 120 min. Quantitative analysis of the band intensities revealed a dose dependent increase in HIF-1a expression in the ischemic vessels. HIF-1a expression level was elevated by 1.5, 2.6 and 3.3 fold after 30, 120 and 240 min of ischemia treatments respectively. However, no significant increase in HIF-1a expression was observed in the ischemia-treated tissues ligated for 10 min (Fig. 8A). This data suggested that partial ischemia was successfully improvised in the chick embryo vascular bed using the strategy of right vitelline artery ligation model. Ranolazine (RL) treatment attenuated HIF-1a expression and protected the free glutathione (GSH) levels in ischemic vessels RL, a well known anti-ischemic drug was used in the present study to recover ischemia-dependent tissue injury. Free glutathi- one (GSH) being the quencher of cellular ROS protects the cells and tissue from ROS mediated damage. In the present study, we measured the level of GSH using CMFDA fluorescence probe. The result depicted that ischemia attenuated the cellular GSH level by 53% while RL administration recovered the GSH level by 52% in the ischemic vessels of chick embryo (Fig. 9A,9B). The expression level of HIF-1a was measured in the ischemic vessels under RL treatment using quantitative RT-PCR technique. Ischemia elevated the level of HIF-1a expression by 38% while Chick Embryo Ischemia Model RL administration attenuated HIF-1a expression in ischemic vessels by 48% (Fig. 8B). However, RL did not affect the expression level of HIF-1a and level of GSH under non-ischemic condition (Fig. 9A,9B). This data elaborated that RL administra- tion protected chick embryo vessels from ischemia induced damage. Partial ischemia interferes with cellular morphology of EC grown on the ischemic bed Morphological analysis of EC grown on the ischemic bed was carried out to evaluate the effect of ischemia on cellular health. EC grown on collagen-coated cover slips were treated under partial ischemia by incubating it on the top of ischemic vascular bed. Images showed a higher number of round cells under ischemic condition. This data demonstrated that ischemic vascular bed driven secondary ischemia in EC interfered with cellular morphology (Fig. 10A). To verify the effect of withdrawing ischemia on cellular morphology, we studied the morphology of the cells by placing the ischemia treated cells on non-ischemic vascular bed. Ischemia reduced the number of normal cells by 32% while placing back the EC on a non-ischemic bed reversed back the number of round cells by 19% (Fig. 10B). This data suggested that ischemia dependent cellular damage can be partially reversed by with- drawing ischemic stress. Ischemic vascular bed driven secondary ischemia compromised cellular viability and induced cellular apoptosis Analysis of apoptosis in EAhy926 cells under partial ischemia treatment was carried out using annexin V-FITC apoptosis detection kit. Result depicted that partial ischemia treatment is apoptotic to cells thus facilitating the binding of annexin V- FITC to the outer part of the plasma membrane (Fig. 10C). Partial ischemia induced endothelial apoptosis by 8 fold compared to non-ischemic control (Fig. 10C). Cellular viability of EC under partial ischemia treatment was assayed using trypan blue dye. Images of the cells revealed that partial ischemia increased the number of trypan blue stained cells by 8 fold (Fig. 10D). Ischemic vascular bed elevated ROS generation in EC Two different ROS parameters were studied in EC under partial ischemia treatment. Superoxide and lipid peroxidation level in cells were measured after incubating EC on ischemic vascular bed using nitro blue tetrazolium (NBT) and TBA protocols respectively. Partial ischemia promoted superoxide generation in EC by 27% while the level of lipid peroxidation under partial ischemia treatment was elevated by 40% compared to non-ischemic control (Fig. 11A,11B). This data suggested that ischemic vascular bed improvised secondary ischemia in EC that in turn elevated ROS level in EC. Partial ischemia reduced the GSH concentration in EC The level of free thiol in EC was measured using CMFDA fluorescent probe. Partial ischemia reduced the level of GSH in EC by 32% (Fig. 11C, 11D). Anti-ischemic drug treatments improved the cellular health condition and preserved the GSH level under partial ischemia To evaluate the protecting role of antioxidant and anti-ischemic drug, we performed morphology counting and GSH measurement PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10524