Diabetes augments and inhaled nitric oxide prevents the adverse hemodynamic effects of transfusing syngeneic stored blood in mice

Abstract

Background: Stored red blood cells (RBCs) undergo progressive deleterious functional, biochemical, and structural changes. The mechanisms responsible for the adverse effects of transfusing stored RBCs remain incompletely elucidated.

Study design and methods: Awake wild-type (WT) mice, WT mice fed a high-fat diet (HFD-fed WT) for 4 to 6 weeks, and diabetic (db/db) mice were transfused with syngeneic leukoreduced RBCs or supernatant with or without oxidation (10% of total blood volume) after storage for not more than 24 hours (FRBCs) or 2 weeks (SRBCs). Inhaled nitric oxide (NO) at 80 parts per million was administered to a group of mice transfused with SRBCs. Blood and tissue samples were collected 2 hours after transfusion to measure iron and cytokine levels.

Results: SRBCs had altered RBC morphology and a reduced P(50) . Transfusion of SRBCs into WT or HFD-fed WT mice did not produce systemic hemodynamic changes. In contrast, transfusion of SRBCs or supernatant from SRBCs into db/db mice induced systemic hypertension that was prevented by concurrent inhalation of NO. Infusion of washed SRBCs or oxidized SRBC supernatant into db/db mice did not induce hypertension. Two hours after SRBC transfusion, plasma hemoglobin (Hb), interleukin-6, and serum iron levels were increased.

Conclusion: Transfusion of syngeneic SRBCs or the supernatant from SRBCs produces systemic hypertension and vasoconstriction in db/db mice. It is likely that RBC storage, by causing in vitro hemolysis and posttransfusion hemoglobinemia, produces sustained NO scavenging and vasoconstriction in mice with endothelial dysfunction. Vasoconstriction is prevented by oxidizing the supernatant of SRBCs or breathing NO during SRBC transfusion.

Fig. 1

Storage-induced morphologic, biochemical, and functional changes in murine RBCs in vitro. (A) Stained smears of FRBCs (top panel) and SRBCs (bottom panel); arrowheads in the bottom panel showing increased numbers of abnormally shaped RBCs after 2-week storage. (B) ODC of FRBCs (—) and SRBCs (- - -). P₅₀, defined as the partial pressure of oxygen at which Hb is 50% saturated, was calculated from the ODC. The ODC of SRBCs was left-shifted with a P₅₀ of 21 ± 1 mmHg; in contrast, the P₅₀ of FRBCs was 43 ± 0 mmHg. (C) Comparison of changes in 2,3-DPG levels of FRBCs (n = 5) and SRBCs (n = 5) with P₅₀ of FRBCs (n = 3) and SRBCs (n = 3). (D) FRBC (●; n = 6) or SRBC (□; n = 6) survival was determined by the percentage of GFP-labeled RBCs measured 24 hours after transfusion divided by the percentage of GFP-labeled RBCs at Time 0 in WT mice. *p < 0.01 differs versus FRBCs.

Fig. 2

Serum iron levels 2 hours after transfusion with FRBCs or SRBCs in awake WT (A), HFD-fedWT (B), and db/db (C) mice. Control, no transfusion, n = 4/group; FRBCs, n = 6/group; SRBCs, n = 6/group. *p < 0.05 differs vs. control and FRBCs.

Fig. 3

Plasma levels of IL-6 (A), Hp (B), and Hx (C) at 2 hours after transfusion of FRBCs or SRBCs in awake WT, HFD-fed WT, and db/db mice. Control, no transfusion, n = 4/group; FRBCs, n = 6/group; SRBCs, n = 6/group. *p < 0.05 differs versus control; †p < 0.01 differs versus FRBCs.

Fig. 4

Changes of hepatic HO-1 mRNA levels at 2 hours after transfusion of FRBCs or SRBCs in awake WT (A), HFD-fed WT (B), and db/db (C) mice. Control, no transfusion, n = 4/group; FRBCs, n = 6/group; SRBCs, n = 6/group. *p < 0.05 differs versus control and FRBCs.

Fig. 5

SBP (mmHg) after different types of transfusions (10% estimated blood volume) in awake WT, HFD-fed WT, or db/db mice. (A) Transfusion with FRBCs (●; n = 5) or SRBCs (□; n = 6) in awake WT mice. (B) Transfusion with FRBCs (●; n = 6) or SRBCs (□; n = 6) in awake HFD-fed WT mice. (C) Transfusion with FRBCs (●; n = 9) or SRBCs with (Δ; n = 12) or without (□; n = 9) breathing iNO (Δ; 80 ppm) in awake db/db mice. (D) Transfusion with supernatant from FRBCs (●; SFRBCs, n = 11), supernatant from SRBCs (□; SSRBCs, n = 7), or oxidized SSRBCs (Δ; n = 6) in awake db/db mice. (E) Transfusion with washed FRBCs (●; n = 9) or washed SRBCs (□; n = 6) in awake db/db mice. *p < 0.05 differs versus FRBCs and SRBCs plus iNO.

Fig. 6

Comparison of changes in systemic hemodynamic measurements in anesthetized db/db mice before and after transfusion of supernatant of FRBCs (SFRBCs, n = 6), supernatant of SRBCs (SSRBCs, n = 6), FRBCs (n = 7) or SRBCs (n = 7). (A) Changes in LVESP (mmHg) before and 10 minutes after transfusion. (B) Changes in SVRI (%) before and 10 minutes after transfusion. *p < 0.05 differs versus either supernatant of FRBCs or the FRBC group