Nitric Oxide Synthase 3 Deficiency Does Not Prevent Inflammatory Adverse Effects Of Transfusing Syngeneic Stored Blood In a Murine Model Of Hemorrhagic Shock

Blood transfusion is a lifesaving treatment for hemorrhagic shock. During storage, red blood cells (RBC) undergo progressive deleterious functional, biochemical and structural alterations, which are collectively termed the “storage lesion”. The association between transfusion of blood stored for more than 14 days and adverse clinical outcomes (increased infection, multi-organ failure and mortality) is controversial. Studying mice with hemorrhagic shock, we recently found that resuscitation with blood stored for prolonged periods (SRBC) was associated with worse outcomes than was resuscitation with fresh blood (FRBC). The mechanisms responsible for the adverse effects associated with transfusion of SRBC are incompletely characterized. However, it is known that transfusion of SRBC increases plasma levels of hemoglobin (Hb), which can scavenge vascular nitric oxide (NO). Intravenous infusion of a solution containing cell-free Hb induces systemic hypertension in wild-type (WT) mice, but not in mice that are congenitally deficient in NO synthase 3 (NOS3^-/-). In the present study, we sought to determine if NOS3^-/- mice are protected from the adverse effects associated with resuscitation of hemorrhagic shock with SRBC.

Leukoreduced, packed RBC from WT C57BL6 mice were stored with 14% CPDA-1 anticoagulant at 4°C for either ≤24 h (FRBC) or 2 weeks (SRBC). Mice, of each genotype that were not subjected to hemorrhagic shock or resuscitation, served as control groups. Anesthetized WT mice and NOS3^-/- mice (on a C57BL6 background) were bled to a mean arterial pressure (MAP) of 40 mmHg over 10 min. After 90 min of hemorrhagic shock, mice were resuscitated with FRBC or SRBC. Survival rates for up to 7 days were determined. In addition, blood and tissue samples were collected at 4 h after resuscitation to measure plasma markers of liver and kidney injury, plasma Hb and interleukin 6 (IL-6) levels, tissue IL-6 mRNA levels, and pulmonary myeloperoxidase activity and mRNA levels. All data are expressed as mean±SD.

Baseline MAP under anesthesia was higher in NOS3^-/- mice than in WT mice (111±4 vs 83±5 mmHg; P<0.01). After hemorrhagic shock and resuscitation with either FRBC or SRBC, MAP returned to the respective baseline values in each genotype. Survival rates at one week did not differ between WT or NOS3^-/- mice resuscitated with either FRBC or SRBC. In both genotypes, after hemorrhagic shock, resuscitation with FRBC elevated plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities similarly as compared to a control group (P<0.01). Plasma levels of ALT and AST were greater after resuscitation with SRBC than after resuscitation with FRBC in both genotypes at 4 h (P<0.01). Resuscitation with SRBC, but not FRBC, increased plasma blood urea nitrogen and creatinine levels similarly in WT and NOS3^-/- mice. Plasma Hb levels were greater in mice resuscitated with SRBC than in those resuscitated with FRBC in both genotypes at 4 h after resuscitation (WT: 202±79 vs 5±2 µM, respectively; NOS3^-/-: 240±51 vs 14±3 µM, respectively; P<0.001 for both). At 4 h after resuscitation, plasma IL-6 levels were greater in mice treated with SRBC than in mice treated with FRBC in both genotypes (WT: 0.8±0.1 vs 0.5±0.1 ng/ml, respectively; NOS3^-/-: 0.8±0.1 vs 0.4±0.1 ng/ml; P<0.01 for both). In both WT and NOS3^-/- mice, IL-6 mRNA levels were greater in the liver, kidney, and spleen after resuscitation with SRBC than after resuscitation with FRBC (P<0.01, all values differ SRBC vs FRBC for both genotypes). Pulmonary myeloperoxidase activity and mRNA levels were greater in both genotypes after resuscitation with SRBC than mice resuscitated with FRBC (P<0.01, all values differ SRBC vs FRBC for both genotypes).

Survival rate after hemorrhagic shock did not differ in WT and NOS3^-/- mice resuscitated with either FRBC or SRBC. Resuscitation with SRBC induced greater tissue injury and a more marked inflammatory response than did resuscitation with FRBC, but there was no difference between the genotypes. Our data suggest that mice with NOS3 deficiency are not protected from the adverse effects associated with resuscitation of hemorrhagic shock with SRBC. These findings suggest that the adverse effects of transfusing blood stored for prolonged periods in mice with hemorrhagic shock are not exclusively attributable to scavenging of NOS3-generated NO by increased plasma Hb levels.

No relevant conflicts of interest to declare.