Cardiopulmonary bypass in the newborn: effects of circulatory cell-free hemoglobin and hyperoxia evaluated in a novel rat pup model

BACKGROUND Infants with congenital heart defects (CHD) are at risk for white matter brain injury. This novel rat pup model characterizes the systemic effects of intravasal cell-free hemoglobin and hyperoxia, hypothesizing that immature endogenous scavenging systems relate to increased vulnerability to conditions present during cardiopulmonary bypass (CPB). METHODS Plasma pharmacokinetics of cell-free human hemoglobin (Hb) was determined after intraperitoneal (i.p.) administration in postnatal day 6 (P6) rat pups. Cell-free hemoglobin degradation, scavenger- and oxidative stress responses in altered oxygen environments were evaluated in P6 rat pups exposed to i.p. cell-free Hb or vehicle and subjected to hyperoxia or normoxia for 24 h. Plasma and liver were analyzed for free heme, haptoglobin, hemopexin, heme-oxygenase 1, and 8-OHdG at 3-120 h post-injection. Baseline scavenging properties were evaluated in P0-P12 rat pups. RESULTS Cell-free Hb displayed peak plasma concentrations of 3.6 ± 0.5 mg/mL (mean ± SD) at 3 h post-administration. Animals exposed to cell-free Hb demonstrated a 30-fold increase in plasma haptoglobin and a decrease in plasma hemopexin to 1/6 of concentrations observed in pups exposed to vehicle. Exposure to cell-free Hb and hyperoxia mediated increased plasma concentrations of free heme (72.7 ± 19.5 μM, mean ± SD) compared to exposure to cell-free Hb and normoxia (49.3 ± 13.1 μM) at 3 h, and an elevated hepatic mRNA expression of heme-oxygenase 1. mRNA expression of haptoglobin and hemopexin was increased in animals exposed to hemoglobin with a mitigated response in pups exposed to hemoglobin and hyperoxia. Animals exposed to hyperoxia displayed an increase in hepatic transcription of scavenger proteins at 24 h. Combined exposure to cell-free Hb and hyperoxia mediated an increased DNA-oxidation at 6 h, whereas all insults conveyed a decrease in DNA-oxidation at 120 h. CONCLUSIONS In this study, we present a novel rat pup model with scavenging characteristics and brain maturation similar to newborns with CHD. We have confirmed a distinct scavenger response after exposure to systemic cell-free hemoglobin. We have indications of an accelerated metabolism of cell-free Hb and of an altered transcription of scavenger proteins in a hyperoxic environment. We believe that this model will prove valuable in future delineation of inflammatory and oxidative end-organ damage following CPB.