Prevention of Pulmonary Hypertension and Suppression of Pulmonary Inflammation and Platelet Activation by Breathing Nitric Oxide in Lambs Subjected to Hemorrhagic Shock and Autologous Stored Blood Transfusion
Authors: Baron David M. , Beloiartsev Arkadi , Nakagawa Akito , Stowell Christopher P. , Bloch Kenneth D. , Zapol Warren M. 
Published: October 2019

Abstract
Abstract 841

Introduction:
Transfusion of red blood cells (RBCs) restores the oxygen carrying capacity of blood after hemorrhagic shock (HS). During extended storage, RBCs undergo functional changes, altering their physiological properties. Recent studies have suggested that transfusion of RBCs stored for prolonged periods of time is associated with increased morbidity and mortality in severely hemorrhaged patients.
Our group has reported a model for autologous transfusion of stored RBCs in lambs and observed that transfusion of RBCs stored for 40 days transiently increased pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in healthy lambs. We hypothesized that HS would sensitize lambs to the adverse effects of stored RBC transfusion, inducing severe pulmonary vasoconstriction, inflammation, and platelet activation. We further hypothesized that inhalation of 80 ppm nitric oxide (NO) during and after transfusion of stored RBCs would prevent pulmonary vasoconstriction and reduce pulmonary inflammation and platelet activation.

Methods:
We studied 3 to 4 month old Polypay lambs weighing 31±1 kg (all data mean±SEM). A tracheostomy and carotid and pulmonary artery catheters were placed under brief isoflurane anesthesia. Lambs were subjected to class 4 HS by withdrawing 50% of their blood volume. Mean arterial pressure was maintained at 50 mmHg for 2 h. Animals were then resuscitated over 1 h with 600 ml packed RBCs (hematocrit 55–60%) and an equal volume of lactated Ringer's solution. One group of lambs (n=6) was resuscitated with the RBCs withdrawn to induce HS (fresh red blood cells, FRBCs). A second group of lambs (n=6) received autologous, leukoreduced RBCs stored in AS-1 additive solution for 39±2 days (stored red blood cells, SRBCs). An additional group of lambs receiving SRBCs (n=5) inhaled 80 ppm NO during and for 21 h after transfusion. Hemodynamic parameters were measured continuously for 24 h.
Plasma hemoglobin concentrations were determined before and after transfusion using spectral deconvolution. Myeloperoxidase enzyme activity was measured in lung tissue samples harvested 21 h after the end of transfusion.
Platelets were isolated from blood withdrawn before induction and immediately after resuscitation of HS. Platelet activation was assessed by measuring the number of P-selectin (CD62P)-expressing platelets with flow cytometry, both in naïve platelets and in platelets stimulated with increasing concentrations of adenosine diphosphate (ADP) ex vivo.

Results:
At the end of transfusion, PVR (253±43 vs. 104±5 dyn·s·cm−5, p=0.01) and mean PAP (24±4 vs. 14±2 mmHg, p<0.001) were higher in lambs transfused with SRBCs than in those transfused with FRBCs. Concurrent inhalation of NO completely prevented the increase of PVR (102±10 dyn·s·cm−5) and PAP (13±1 mmHg) induced by transfusing SRBCs. PAP correlated with peak plasma hemoglobin concentrations (R2=0.61, p=0.003). Two of the 6 lambs transfused with SRBCs developed severe systemic hypoxemia and pulmonary edema, whereas none of the lambs resuscitated with FRBCs or SRBCs during concurrent inhalation of NO developed hypoxemia or symptoms of respiratory compromise.
Pulmonary myeloperoxidase enzyme activity of lambs transfused with SRBCs (11±2 U/g) was higher than that of lambs resuscitated with FRBCs (4±1 U/g, p=0.007), but was not elevated when animals breathed NO (8±1 U/g, p=0.09).
There was no difference in the percentage of CD62P-expressing platelets after resuscitation of HS with either FRBCs or SRBCs (1.4±0.3 vs. 1.7±0.3 %, p=0.25). However, when stimulated with increasing concentrations of ADP, more platelets from lambs transfused with SRBCs expressed CD62P than those from lambs transfused with FRBCs. This increase in CD62P-expressing platelets after transfusion of SRBCs was attenuated by breathing NO.

Conclusions:
Hemorrhagic shock sensitizes lambs to the transfusion of stored autologous RBCs. Resuscitation of 6 lambs with severe HS by transfusion of SRBCs caused marked pulmonary vasoconstriction and inflammation, as well as severe hypoxemia and pulmonary edema in 2 lambs. Inhalation of NO completely prevented pulmonary vasoconstriction and attenuated pulmonary inflammation and respiratory failure. Platelet activation was not altered by transfusion of SRBCs, but ADP more readily activated these platelets. Therefore, patients with HS might benefit from breathing NO when resuscitated with SRBCs.

Disclosures:
Bloch: Massachusetts General Hopsital: Research Funding. Zapol:Massachusetts General Hopsital: Dr. Zapol receives royalties from patents on inhaled nitric oxide licensed by MGH to Linde (Munich, Germany) and Ikaria (Clinton, NJ)., Dr. Zapol receives royalties from patents on inhaled nitric oxide licensed by MGH to Linde (Munich, Germany) and Ikaria (Clinton, NJ). Patents & Royalties.