Chick Embryo Ischemia Model Figure 9. HIF-1a expression level in ischemic vitelline vascular tissue. (A) A time dependent HIF-1a expression in the ischemic tissues were quantified using quantitative RT-PCR technique. After treatment mRNA was isolated from the tissues using RNA extraction kit followed by quantitative RT-PCR. Total extracted RNA was quantified by running a 2%agarose gel containing ethidium bromide. Total RNA loading for cDNA conversion was then normalized from the band luminosity of total RNA from different samples. Amplified products using specific primers for HIF-1a was then run on 2% agarose gel and visualized. The band luminosity was then quantified and plotted. Band intensities were considered as directly proportional to the HIF-1a mRNA level in the tissues. Images are the representative of 3 individual experiments. *P = 0.012 and **P,0.001 versus 0 min time point. (n = 3) (B) HIF-1a expression level was measured in ischemic vascular bed with RL treatment. RL was treated during the period of ischemia. RL attenuated ischemia induced HIF-1a expression. Images are the representative of 3 individual experiments. **P,0.001 versus non- ischemia and yyP,0.001 versus ischemia. (n = 3). doi:10.1371/journal.pone.0010524.g009 damage. Similarly, in the present study, we observed a higher level of superoxide generation in both ischemic tissues and in cells treated with the ischemic vascular bed. However, we observed that blocking of right vitelline artery suppressed hydrogen peroxide production. Hydrogen peroxide is known to confer protection to ischemic tissues. The work of Yada et al. (2006) showed that endogenous hydrogen peroxide, in cooperation with NO, plays an important cardioprotec- tive role in coronary ischemia reperfusion injury in vivo [30]. It has been reported that hypoxia activates catalase pool in the cells. A classic work by Oshino et al. (1975) demonstrated that the rate of hydrogen peroxide production in the ‘‘haemoglobin-free’’, ‘‘non- circulatory’’ perfused liver of rats decreased to almost half in the livers of starved and phenobarbital-pretreated rats [31]. Enhanced superoxide generation and simultaneous fall in hydrogen peroxide in the present partial ischemia model indicates a reduced dismutation of superoxide by its classical SOD pathway, which is the prime source of hydrogen peroxide. In stead superoxide possibly interacts with NO to form peroxynitrite that is evident from the results of the present study. Work of Scaduto et al., 1988 depicted that the content of GSH in the rat declined to 40% of control values during 35 min of renal artery occlusion [32]. In the present partial ischemia model, the level of GSH, a potent quencher of ROS was suppressed. We deem that low GSH level under partial ischemia leads to higher ROS generation that caused damage to EC and cardiomyofibroblast cells as observed in the present study. The result of present work further ascertains that deteriorating functional parameters of the EC is the combinational effect of ischemia and associated ROS generation from the ischemic vascular bed. RL, a specific inhibitor of late sodium current reduces sodium overload and hence ameliorates disturbed ion homeostasis in cardiac cells [33–39]. In the present study we administered RL as an anti-ischemic drug and evaluated its activity on different functional and biochemical parameters of the cells and tissues under ischemia. As observed by different groups, present study also suggests that RL can protect the cells and tissues from secondary ischemia developed by incubating the cells on ischemic vascular bed of chick embryo. The results offered another line of support that the present model is capable of inducing ischemia in cells and tissues. Reports from earlier findings described the protective role of NAC after ischemia or ischemic reperfusion injury [40,41]. In addition to RL, we used these two well known anti-ischemic drugs NAC and TRZ [42,43] to elucidate the efficacy of these drugs in recovering ischemia based cellular injury in the present model. NAC and TRZ both protected EC from ischemic vascular bed driven secondary ischemia. PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10524