Gelfoam angiogenesis assay These experiments were approved by the MD Anderson Cancer Center Institutional Animal Care and Usage Committee

ther, kept at the target MAP (35 mmHg) for an additional 60 minutes. Hearts were immediately harvested and snap frozen in liquid nitrogen. Nucleosome levels were measured in protein extracts of frozen sections of the heart and the results corrected for total protein and plotted as a function of the duration of the hypotensive period. Nucleosome levels increased exponentially with duration of hypotension (Pearson correlation coefficient = 0.764, p,0.0001, UHS group not included in the analysis)nucleosome ELISA assays (Figure 3A). Nucleosome levels in hearts from SBR50/IL-6 rats were reduced 94% compared to placebotreated SBR50 rats (p,0.05, one-way ANOVA with StudentNewman-Keuls test). TUNEL assays of sections of rat hearts confirmed these findings (Figure 3B and 3C).Mortality following initially successful As a normal, Western blot analysis Western blot analysis was performed by typical techniques resuscitation from severe HS results from multi-organ dysfunction or failure [4,5]. Following resuscitation, all SBR50 rats died regardless of whether or not they received IL-6 suggesting that the 50% SBR protocol caused failure of one or more vital organ(s) other than the heart that was not reversed by IL-6. To investigate the impact of IL-6 on mortality after trauma plus hemorrhage, SBR35 rats, which also experienced HCC and cardiomyocyte apoptosis (Table 1 and Figure 2), were randomized to receive either placebo or IL-6 (10 mg/kg) at the start of resuscitation (Figure 5). Similar to the effect of IL-6 treatment on HCC in the SBR50 group, all animals in the SBR35/IL-6 group were successfully resuscitated compared to 80% of rats in the placebo-treated SBR35 group (data not shown). Furthermore, mortality was reduced nearly 5 fold from 72% in placebo-treated SBR35 rats to 15.4% in SBR35/IL-6 group (p,0.005, Kaplan-Meier survival analysis).IL-6 activates Stat3, which has previously been demonstrated to activate the transcription of several anti-apoptotic genes and to contribute to apoptosis resistance in cancer cells (reviewed in [21]). To assess if the anti-apoptotic effect of IL-6 is mediated by Stat3 activation, we first determined if Stat3 is activated in the hearts of rats resuscitated with IL-6. Extracts of cryotome sections of the heart harvested 1 hour after IL-6 administration were examined by EMSA using radiolabeled duplex hSIE (Figure 6). Stat3containing gel-shift bands, SIF-A and SIF-B (Stat3 homodimers and Stat3:Stat1 heterodimers, respectively) were more readily detected in heart extracts from SBR50/IL-6 rats than in heart extracts from placebo-treated SBR50 rats. Phosphoimaging analysis of Stat3-containing bands indicated that Stat3 DNAbinding activity is increased over two fold in the hearts of SBR50/ IL-6 rats compared to placebo-treated SBR50 rats (p = 0.028, Student's t-test). Levels of phosphorylated Stat3 within hearts of SBR50/IL-6 rats also were increased, which confirmed the EMSA results (data not shown). To determine if activation of Stat3 downstream of IL-6 was important for the improved resuscitation success observed in SBR50/IL-6 rats, we pre-treated rats 24 hr before SBR50 and IL6 resuscitation with a G-rich, quartet forming oligodeoxynucleotide (GQ-ODN, T40214) that specifically binds to and inhibits the activity of Stat3 [9] or with a non-specific oligodeoxynucleotide (NS-ODN).