Alterations in gene expression after induction of profound hypothermia for the treatment of lethal hemorrhage.

INTRODUCTION

We have previously demonstrated that induction of profound hypothermia improves long-term survival in animal models of complex injuries/lethal hemorrhage. However, the precise mechanisms have not been well defined. The aim of this high-throughput study was to investigate the impact of profound hypothermia on gene expression profiles.

METHODS

Wistar-Kyoto rats underwent 40% blood volume arterial hemorrhage over 10 minutes and were randomized into two groups based on core body temperatures (n = 7 per group): hypothermia (H, 15 degrees C) and normothermia (N, 37 degrees C). Hypothermia was induced by infusing cold isotonic solution using a cardiopulmonary bypass (CPB) setup. After reaching target body temperature, low-flow state (CPB flow rate of 20 mL x kg x min) was maintained for 60 minutes. Hypothermic rats were rewarmed to baseline temperature, and all rats were resuscitated on CPB and monitored for 3 hours. The N group underwent identical CPB management. Sham rats (no hemorrhage and no instrumentation) were used as controls. Blood samples were collected serially, and hepatic tissues were harvested after 3 hours. Affymatrix Rat Gene 1.0 ST Array (27,342 genes, >700,000 probes) was used to determine gene expression profiles (n = 3 per group), which were further analyzed using GeneSpring (Agilent Technologies, Santa Clara, CA) and GenePattern (Broad Institute, Cambridge, MA) programs. Data were further queried using network analysis tools including Gene Ontology, and Ingenuity Pathway Analysis (Ingenuity Systems). Key findings were verified using real-time polymerase chain reaction and Western blots.

RESULTS

Induction of hypothermia significantly (p < 0.05) decreased the magnitude of lactic acidosis and increased the survival rates (100% vs. 0% in normothermia group). Five hundred seventy-one of 23,000 genes had altered expression in response to the induction of hypothermia: 382 were up-regulated and 187 were down-regulated. Twelve key pathways were specifically modulated by hypothermia. Interleukin-6, interleukin-10, p38 mitogen-activated protein kinase (MAPK), nuclear factor kappa-light-chain-enhancer of activated B cells, glucocorticoids, and other signaling pathways involved with acute phase reactants were up-regulated. Multiple metabolic pathways were down- regulated. The largest change was in the peroxisome proliferator-activated receptor gamma gene that codes for a transcriptional coactivator, which in turn controls mitochondrial biogenesis, glycerolipid, and other metabolic pathways in the liver. Apoptotic cell death cascades were activated in response to blood loss (H and N groups), but multiple specific anti-apoptotic genes (baculoviral Inhibitor of apoptosis protein repeat-containing 3, BCL3L1, NFKB2) displayed an increased expression specifically in the hypothermia treated animals, suggesting an overall pro-survival phenotype.

CONCLUSIONS

Profound hypothermia increases survival in a rodent model of hemorrhagic shock. In addition to decreasing tissue oxygen consumption, induction of hypothermia directly alters the expression profiles of key genes, with an overall up-regulation of pro-survival pathways and a down- regulation of metabolic pathways.