Chick Embryo Ischemia Model Figure 11. Level of different reactive oxygen species and GSH in ischemic EC. (A) Level of lipid peroxidation in ischemic vascular bed treated EC was quantified using TBA protocol. EC were treated under ischemia for 2 h followed by processing as mentioned in the materials and methodssection. Data were normalized by counting the number of cells using hemocytometer. Elevated lipid peroxidation was noted under ischemia treatment. *P = 0.012 versus non-ischemic. (n = 5) (B) Superoxide production by ischemia treated EC was measured using NBT. Cells were treated under ischemia for 2 h followed by incubation with NBT for another 2 h. The formed formazen dye was extracted from the cells as mentioned in methodology. Data were normalized by counting the number of cells using hemocytometer. **P,0.001 versus non-ischemic. (n = 5) (C) Ischemia treated EC were probed with CMFDA to measure the level of GSH in the cells. Cells were treated with ischemic vascular bed driven secondary ischemia for 2 h followed by probing with CMFDA for 30 min. Images were acquired by DP71 camera adapted to an Olympus IX71 microscope. Images are the representative of 5 individual experiments. (D) The green luminosity from the CMFDA probed fluorescent images were measured using Adobe Photoshop version 7.0. Green luminosity of 40 different cells from 5 different sets of experiment were measured and plotted. Ischemia reduced the level of free thiol in EC. **P,0.001 versus non-ischemic. (n = 5). doi:10.1371/journal.pone.0010524.g011 specified rhythm specifically in a defined vessel in the vascular bed by using macro-manipulator to achieve the intermittent and modulated ischemia along with reperfusion effects. The results of the present work also suggested that anti- ischemic efficacy of any drug can be evaluated in the present model before studying the drug in any other animal models. Therefore, the present model can be used as an additional model along with other in vivo ischemia models to reduce animal experimentations. Materials and Methods Materials Dulbecco’s modified Eagle’s medium (DMEM) was purchased from PAN-Biotech GmbH, Am Gewerbepark, Aidenbach. Fetal bovine serum was from Invitrogen Life technologies. Diamino fluorescein diacetate, 5-chloromethylfluorescein diacetate, amplex red, dihydrorhodamine 123 (DHR) was purchased from Moleculer Probe, Oregon, USA. RNA isolation spin prep kit was from Medox Inc., India. cDNA Mulv reverse transcriptase and taq polymerase was purchased from New-England Biolabs, MA and Finnzymes, Espoo, Finland respectively. RL [N-(2,6-dimethylphe- nyl)-2-[4-[2-hydroxy-3-(2-methoxyphenoxy) propyl]piperazin-1- yl]acetamide] and TRZ was purchased from Sigma Chemicals, St. Louis, MO, USA. Nitro blue tetrazolium and NAC was purchased from Hi-Media, Bangalore, India. All other chemicals were of reagent grade and were obtained commercially. Cell culture Immortalized endothelial hybrid cell lines, EAhy926 were gifted from Dr C.J.S. Edgell. H9C2 myofibroblast cells were purchased from NCCS, Pune, India. Both the cells were maintained and cultured in DMEM supplemented with 10% FBS (v/v) and 1% penicillin (w/v) and streptomycin (w/v). Animals Fertilized white Leghorn chick (Gallus domesticus L.) eggs were obtained from Poultry Research Station, Nandanam, Chennai, TN, India and incubated at 37–38uC, with the blunt end up and at a relative humidity of 70–80%. Caprine heart samples were collected from the government authorized slaughter houses. Creating partial ischemia on chick embryo vascular bed using artery ligation model In the early stages of chick embryo development, two distinct circulatory systems are established in an embryonic system, one for the embryo itself maintaining the blood circulation inside the PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10524