The Effect Of Increasing Nitrie Oxide Bioavallability On The Adverse Effects Of Stored Blood Transfusian Into Mice

【Background】Red blood cell (RBC) transfusion is a life-saving therapyemployed to treat patients with anemia or major blood loss (e.g. hemorrhagicshock). By replacing blood hemoglobin (Hb), red cells sustain tissue and organoxygenation. Every year, about 80 million units of blood are collected worldwide.In the United States, about 15 million units of RBCs are transfused annually, withan estimated mean duration of storage before transfusion of 17 days. During exvivo storage, RBCs undergo numerous biochemical, structural and functionalalternations, which are collectively termed the"storage lesion". Red celltransfusion is an empirical treatment, which has never undergone prospectiverandomized testing in the fashion of that of a new drug. Accumulating evidencehas been reported demonstrating an association between the duration of RBCstorage and deleterious clinical outcomes. A number of clinical studies havedocumented that transfusion of blood stored for over 14 days is associated withan increased rate of infection, mortality, multi-organ failure, and length of stay inhospital in selected patient populations including those undergoing cardiac surgical procedures, multiple trauma victims, and critically ill patients.The precise mechanisms responsible for the deleterious effects on the hostproduced by SRBC transfusion are not fully understood. Substantial hemolysisincreases as a part of the storage lesion with prolonged RBC storage. Given theacceptable RBC viability (75% post-transfusion recovery) in current FDAguidelines, it is estimated that approximately 25% of red cells will bedecomposed and cleared out from recipients'circulation within the first 24 hoursresult in considerable intravascular hemolysis, particulary in the setting ofmassive transfusion. Even a low level of intravascular hemolysis severely disruptnitric oxide (NO) bioavailability at the endothelium, via accelerated NOdioxygenation reactions with extracellular free hemoglobin (Hb). This processcontributes to endothelial dysfunction, adhension molecule expression, plateletand hemostatic activation, and reactive oxygen species (ROS) generation. On theother hand, RBC possess NO-generating function through nitrite reduction bydeoxyHb and red cell endothelial NO synthase (NOS) activity. During ex vivostorage, the NO production is dramatically destructed. We hypothesize that theRBC storage is associated with imbalance between NO production and NOdestruction, which results in a decrease of intravascular NO bioavailability. Thedecreased NO bioavailability may be responsible for stored RBC transfusionassociated deleterious effects.The present study was designed to investigate the role of nitric oxidebioavailability on the adverse effects of stored blood transfusion into mice, and toexplore the possibility of supplementation with exogenous NO in preventing andreducing the adverse effects associated with stored RBC transfusion. Experiment 1 Investigation of storage lesion in murine stored red blood cells【ObjectivObjective】To investigate storage lesion of murine stored RBC and set up amurine RBC storage system.【Methods】Blood was withdrawn aseptically by open chest cardiac puncturefrom C57BL/6J WT mice into a syringe containing CPDA-1. The finalconcentration of CPDA-1 was 14%. After collection, blood was leukoreduced bypassage through a neonatal leukoreduction filter. Leukoreduced blood wascentrifuged for 15 min at 600 g, and adjusted to a hematocrit (Hct) level of70-75% by partially removing plamsa, and stored in Eppendorf tubes at 4°C.RBCs were divided into two groups: RBCs in fresh RBC group (FRBC) werestored for less than 24 hours while the RBCs in stored RBC group (SRBC) werestored for 2 weeks. The physiological and biochemical parameters, and structuraland functional alternations were compared between these two groups.【Results】We found pHa, HCO3-, and PaO2 were significantly decreased, andPaCO2, SBE, blood urea nitrogen (BUN), and lactate were increased in SRBCwhen compared to that in FRBC. In addition, we observed that extracellularsodium level decreased with an elevation of potassium level in SRBC supernatant.The hemolysis level of SRBC was 8 times higher than that of FRBC. Accordingly,we observed an increase of the supernatant Hb and metHb level of SRBC ascompared those of FRBC. FRBC and SRBC were smeared and stained.Discocytes dominated the cell population in FRBC. After 2 weeks of storage, themajority of SRBC red cells are abnormally shaped, e.g. echinocytes,spheroechinocytes, stomatocytes, spherostomatocytes, and spherocytes, etc. TheODC of SRBC was left-shifted with a decreased P50. We observed that RBC2,3-DPG and ATP levels markedly decreased in SRBC. To measure the lifespanof transfused RBCs, we measured RBC survival using GFP labeled erythrocytes. The transfused SRBCs experienced rapid clearance within the first 24 h followedby an extended survival to 8 days. The RBC decay slope of SRBC wassignificantly different from that of FRBC within the first 24 h, but was similar toFRBC thereafter. The mean 24 h recovery of transfused GFP labeled RBCs wasapproximately 99% in FRBC and 68% in SRBC. TXB2 concentration wassignificantly elevated in the supernatant of SRBC after 2 wks of storage.【ConclusiConclusion】Murine RBCs experienced significant biochemical,morphological and functional changes after 2 wks of storage.Experiment 2 The effect of a decrease of NO bioavailability on stored RBCtransfusion and the underlying mechanisms【Objective】To investigate the effect of stored RBC transfusion on mice with orwithout endothelial dysfunction, and to explore the role of NO bioavailability inthis process.【Methods】Fresh or stored RBCs were transfused (10% of estimated bloodvolume) via a tail vein into either C57BL/6 (WT) mice, WT mice fed with highfat diet (HFD) or diabetic db/db mice. Three groups of awake WT, HFD ordb/db mice were studied. One group was control group; a second and third groupwas transfused with either FRBC or SRBC. Additional six groups were added indb/db mice. A fourth group of db/db mice received SRBC and breathed 80 ppmNO in air from 10 min prior to transfusion until sacrifice at 2 h after transfusion.The other four groups received supernatant of FRBC, supernatant of SRBC,washed SRBC or washed FRBC, respectively. The remaining group receivedoxidized supernatant of SRBC. Systolic blood pressure (SBP) was measured witha noninvasive blood pressure system. Some mice were sacrifice at 10 min after transfusion for physiological and biochemical parameters measurement. Theother mice were sacrificed 2 h after transfusion. Heparinized plasma or serumsamples were then obtained for determination of iron, interleukin-6 (IL-6). Liverspecimens were snap-frozen in liquid nitrogen for subsequent determination oftissue iron and mRNA levels of various cytokines.【ResultResults】In WT mice, transfusion of either FRBC or SRBC significantlyelevated the PaO2, Na+, BUN, Hct, total Hb, and plasma Hb level. We observedthat blood lactate level was only increased after SRBC transfusion groups. Asexpected, the extracellular Hb level was significantly higher in plasma of theSRBC groups (both WT and db/db) than that in the FRBC or control group. Indb/db mice at 10 min after transfusion of either FRBC or SRBC, there was anincreased Hct, total Hb, and plasma Hb. Two hours after transfusion of eitherFRBC or SRBC, Hct and total Hb levels were also increased in WT, HFD, anddb/db mice. Transfusion of SRBC caused a significant increase of plasma Hblevels in all three types of mice. Transfusion of either FRBC or SRBC did notchange systemic blood pressure SBP in awake WT or HFD mice. In contrast,transfusion of SRBC but not FRBC caused immediate systemic hypertension indb/db mice. Breathing 80ppm NO completely prevented the systemichypertension induced by the transfusion of SRBC in db/db mice. In addition,transfusion of the supernatant from SRBC but not washed stored cells producedimmediate systemic hypertension in db/db mice. Fresh supernatant infusion didnot produce systemic hypertension. Oxidation free Hb into metHb blocked thehypertensive reaction of supernatant of SRBC transfusion into db/db mice. Serumiron levels significantly increased only after transfusion of SRBC but not FRBCin WT, HFD, and db/db mice. We did not find any increase of hepatic iron levelsby 2h after transfusion in all three groups. Transfusion of SRBC induced an elevated plasma IL-6 level and hepatic IL-6 mRNA expression in WT, HFD, anddb/db mice. Hepatic HO-1 mRNA levels significantly increased after atransfusion of SRBC but not FRBC in WT, HFD, and db/db mice. Hepatichepcidin mRNA levels were significantly increased after transfusion of SRBC butnot FRBC in both WT and HFD mice, but not remarkably elevated in db/db mice.【ConclusiConclusion】Transfusion of stored RBC was associated increasedinflammatory responses in WT, HFD and db/db mice. The vasoconstrictive effectof transfusing SRBC into db/db mice was resulted from NO scavenging byextracellular free Hb. Supplementation of exogenous NO completely abatedtransfusion of stored RBC induced hypertension.Experiment 3 Role of NO bioavailability on the adverse effects ofResuscitation with SRBC into mice after hemorrhagic shock【Objective】TO investigate the effect of SRBC resuscitation in mice afterhemorrhagic shock, and to explore the role of NO bioavailability decreasing onthe adverse effects of SRBC transfusion.【Methods】Sixty WT and HFD mice were randomly divided into three groups. Asham control group experienced the similiar anesthesia, mechanical ventilationand surgical procedures but without inducing hemorrhagic shock. A second andthird mouse group was resuscitated with either FRBC or SRBC followinghemorrhagic shock, respectively. After 2 h, mice selected for survival studieswere extubated, allowed to breathe at FiO2=0.21, and kept warm until theyrecovered. An additional group of 20 HFD mice was resuscitated with SRBC, butthis group breathed 80 ppm NO from 10 min before blood and fluid resuscitationand until 2 h after fluid resuscitation was completed. Survival rates after hemorrhagic shock and resuscitation were compared within and between the WTand HFD groups. Additional mice were enthanized at 2 h after resuscitation.Blood samples were obtained by heart puncture for chemical analysis. Plasmasampes were obtained to measure IL-6, haptoglobin (Hp), hemopexin (Hx),aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinephosphokinase (CPK). Organs (lung and liver) were snap-frozen for storage inliquid nitrogen and subsequent determination of cytokine mRNA expressionlevels.【ResultResults】Compared to WT mice, the baseline MAP was significantly higher inHFD mice. MAP was not different between WT and HFD mice resuscitated withFRBC, SRBC or SRBC plus 80 ppm inhaled NO. We found no short-term (12hours) or long-term (7 days) survival difference between WT mice resuscitatedwith either FRBC or SRBC (similar Kaplan-Meier survival curves). However,SRBC resuscitation was associated with reduced long-term survival rates in HFDmice as compared to WT mice (0% vs 40%, P <0.05). In addition, in HFD mice,resuscitation with SRBC significantly decreased both short and long-termsurvival as compared to resuscitation with FRBC. NO inhalation significantlyimproved the short-term survival rates of HFD mice (80% survival with NObreathing versus 30% survival without NO, P=0.014). However, there was nolong-term survival benefit over SRBC resuscitation produced by NO inhalation(P=0.093). No differences in arterial blood gas tensions or pHa was measuredafter resuscitation with either FRBC or SRBC. However, a markedly increasedlactate level was found in HFD mice resuscitated with SRBC as compared toFRBC resuscitation. The elevated lactate level after SRBC resuscitation in HFDmice was significantly reduced by inhalation of NO. At 2 h after resuscitation,darkening of the splenic color and a significantly increased spleen weight were measured in WT mice resuscitated with SRBC. There was a trend of spleenweights to increase in SRBC resuscitated (with or without NO) HFD mice(P=NS). No differences of any other organ weights (lung, heart, liver, kidneys)were detected between any groups. Compared to control mice, hemorrhagicshock and resuscitation induced a significant increase of AST, BUN and CPK inWT and HFD mice. Plasma AST, BUN and CPK levels in SRBC resuscitated WTor HFD mice were significantly increased compared to the FRBC resuscitatedmice. In HFD mice, SRBC induced higer levels of these organ dysfunctionmarkers as compare to WT mice. Inhaled 80ppm NO significanly reduced AST,BUN and CPK levels in HFD mice. The 2,3-DPG concentrations and P50 in theblood of HFD mice 2 h following resuscitation with SRBC were significantlylower than those of the FRBC group. In both WT and HFD mice, SRBCresuscitation dramatically increased plasma Hb and heme levels. Breathing NOwas associated with a reduction of plasma free Hb levels in HFD miceresuscitated with SRBC. In consistent with plasma Hb and heme changes,plasma levels of both Hp and Hx were significantly decreased in WT miceresuscitated with SRBC as compared to either the FRBC resuscitation group orthe control group. All group of HFD mice with hemorrhagic shock andresuscitation resulted in a decrease of both plasma Hp and Hx as compared to thecontrol group. At 2 hours after resuscitation, plasma IL-6 and tissue IL-6 mRNAexpression levels were significantly increased in all SRBC resuscitation groups,with an exacerbated reaction in HFD mice. Similarity, tissue TNFαand TFmRNA expression were not changed among all of the WT groups, withsignificant elevation of all the HFD mice no matter what fluid was used forresuscitation. SRBC resuscitation increased both HO-1 and hepdicin mRNAexpression levels of both WT and HFD mice. 【ConclusiConclusion】Resuscitation of SRBC in mice after hemorrhagic shock wasassociated with adverse effects, including higher mortality rate, multi-organdysfunction, and systemic inflammatory reaction. Increase NO bioavailability byNO inhalation increased short-term survival via increasing microcirculationperfusion and improving organ function.Conclusions1. After 2 week ex vivo storage, murine RBC experienced significantphysiological, biochemical, morphological and functional alternations.2. Low dose of stored RBC top-load transfusion into WT or HFD mice withendothelial dysfunction was related to inflammatory reactions. Top-loadtransfusion of SRBC into diabetic db/db mice induced inflammatory andvasoconstrictive reactions.3. Resuscitation of SRBC in WT and HFD mice after hemorrhagic shock wasassociated with adverse effects, including higher mortality rate, multi-organdysfunction, and systemic inflammatory reaction.4. HFD and db/db mice with endothelial dysfunction were predisposed toSRBC transfusion induced adverse effects. Transfusion of stored RBC intothese mice induced exaggerated deleterious effects.5. The etiology of SRBC transfusion related deleterious effects wasmultifactorial, in which the decrease of NO bioavailability played animportant role. Supplementation of exogenous NO was not able tocompletely abate, but significantly attenuated some of the adverse effects.