Chick Embryo Ischemia Model Figure 7. Studying the angiogenesis pattern in vessels adjacent to ischemic area. (A) Angiogenesis pattern of the vessels adjacent to the ischemic area were followed for 3 h. The images of the vessels adjacent to the ischemic area were taken with Nikon Cool Pix camera adapted to a stereo microscope. Images were converted to gray scale and presented. Inset showed the magnified images from ischemia group which revealed formation of new vessels in the area. Yellow arrows showed the formation of new vessels in the area. Images are the representative of 5 individual experiments. (B) Analysis of the images were carried out using Angioquant software. Number of separate vessel complexes was analyzed by the software for different time point. Data presented as fold increase with time. **P,0.001 versus respective time point under non-ischemia. (C) Similarly, the software calculated the length of the vessels in different time of incubation. Data presented as fold increase with time. *P,0.05 and **P,0.001 versus respective time point under non-ischemia. (D) The size of the vessels was calculated by the Angioquant software. Data presented as fold increase with time. **P,0.001 versus respective time point under non-ischemia. (E) The number of junctions in the vessels were calculated by the software and plotted. *P,0.05 and **P,0.001 versus respective time point under non-ischemia. doi:10.1371/journal.pone.0010524.g007 experiments in ischemia treated EC in the presence and absence of anti-ischemic drugs. Results depicted that ischemia induced rounding up of EC while RL, N-acetyl cysteine (NAC) and trimetazidine (TRZ) treatments protected cells from ischemia induced stress. However, non-ischemic cells were insensitive to RL, NAC and TRZ treatment (Fig. 12A). GSH measurement in ischemia treated EC using CMFDA elaborated that ischemia reduced the level of GSH while RL, NAC and TRZ potentiate the cells to maintain normal GSH concentration under ischemia (Fig. 12B). However, RL did not show any effect on the level of GSH in non-ischemia treated EC. Morphology of H9C2 cells under ischemia was studied with and without RL administrations. Ischemia promoted the rounding up of H9C2 cells while administration of RL protected the cells from ischemia induced rounding up (Fig. 12C). However, non-ischemic cells were insensitive to RL administration. Partial ischemia induced HIF-1a expression in EC while RL attenuated ischemia induced over expression of HIF-1a To evaluate the protective effect of RL, HIF-1a mRNA expression was measured in RL treated EC under partial ischemia treatments. Quantitative RT-PCR data elaborated that ischemia induced HIF-1a expression in EC by 6 and 11 fold in 30 min and 60 min ischemia treated cells respectively (Fig. 13A). Administra- tion of RL blocked ischemia induced HIF-1a expression by 1.8 fold (Fig. 13B). Ischemic vascular bed mediated partial ischemia in caprine heart elevated the level of creatine phospho kinase-MB (CK-MB) activity in the heart tissues To evaluate the efficacy of the present model on creating secondary ischemia in heart tissues, we incubated the caprine heart PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10524