Liposome-encapsulated Hemoglobin Research Articles

Liposome-encapsulated hemoglobin rescues hemorrhagic shock heart through anti-ischemic and anti-arrhythmogenic effects on myocardium in repetitive 65% bleeding rat model.

Phase I clinical trial of an artificial oxygen carrier (liposome-encapsulated hemoglobin vesicles [HbVs]) is safely completed, considering the other clinical application. Herein, we aimed to investigate the resuscitation effects of HbVs in cases of lethal hemorrhage, including the mechanisms involved. Optical mapping analysis (OMP) and electrophysiological studies (EPS), immunostaining pathological examination for hypoxia-inducible factor-1α (HIF1-alpha) in the heart tissue, and blood troponin I (TnI) level measurements were performed in rats that underwent five rounds of spontaneous arterial bleeding with up to 65% hemorrhage. Ten rats in each group were resuscitated by a transvenous infusion of 5% albumin (ALB), washed erythrocytes (wRBC), HbVs (HbV), 50% HbV diluted by 5% albumin (50% HbV), and 66% HbV diluted by 5% albumin (66% HbV). The rats in the ALB and 50% HbV groups died, whereas those in the other groups survived. OMP showed impaired action potential duration dispersion (APDd) in the left ventricle in the ALB and 50% HbV groups, which was attenuated in the other groups. Lethal arrhythmias were provoked by EPS in the ALB and 50% HbV groups but not in the other groups. HIF1-alpha was positively stained only in the ALB and 50% HbV groups. TnI levels were elevated in the ALB and 50% HbV groups. Acute lethal hemorrhage causes myocardial ischemia with hypoxia and arrhythmias, which may be induced by impaired APDd and myocardial damage, reflected in the increased levels of HIF1-alpha and TnI. HbVs could be useful for resuscitation and may help save patients with injuries such as gunshot wounds.

Abstract 202: Liposome-Encapsulated Hemoglobin (HbV) Rescues Hemorrhagic Shock Heart Through Anti-Ischemic and Anti-Arrhythmogenic Effects on Myocardium in Repetitive 65% Bleeding Rat Model

Phase I clinical trial of Liposome-encapsulated hemoglobin vesicles (HbVs) was successfully accomplished. Since several ameliorating mechanisms are not fully understood on HbVs, to investigate its mechanisms of salutary effect on myocardium, optical mapping analysis (OMP) and electrophysiological study (EPS), immunostaining pathological examination for HIF1-alpha in heart and measurement of blood troponin I (TnI) were performed in 30% spontaneous arterial bleedings five times up to approximately total 65% hemorrhage in rats. Eighteen rats in each group were resuscitated by immediately after bleeding by same volume as bleedings each five times by transvenous infusion of 5%albumin solution (ALB-group), washed erythrocytes (wRBC-group) and HbVs (HbV-group). Survival effects over 24 hours were examined. After excising heart, OMP and EPS were performed. All rats died in ALB-group, whereas all survived in wRBC-group and HbV-group. OMP showed impaired (prolonged) action potential duration dispersion (APDd: which is arrhythmogenic index) in LV in ALB-group, whereas APDds were attenuated in HbV-group and wRBC-group (26±4 vs. 11±6, 14±5ms, P<0.05). Lethal arrhythmias (ventricular tachycardia [VT]/ventricular fibrillation [VF]; VT/VF) were provoked by EPS and/or spontaneously occurred in ALB-group whereas not in both wRBC-group and HbV-group. HIF1-alpha was positively stained and TnI levels were significantly elevated only in ALB-group (Peak TnI; 186±47 vs. 64±25, 91±24, 44±10pg/mL, P<0.05: Figure). Conclusions: HbVs rescue lethal hemorrhage through improving lethal arrhythmias and myocardial damages by reflected by inducing HIF1-alpha and elevated TnI. HbVs could be useful for resuscitation and saving life from gun-shot arterial bleeding on-site occurred in terrorisms.

Red Blood Cell Substitutes: Liposome Encapsulated Hemoglobin and Magnetite Nanoparticle Conjugates as Oxygen Carriers.

The established blood donation and transfusion system has contributed a lot to human health and welfare, but for this system to function properly, it requires a sufficient number of healthy donors, which is not always possible. Pakistan was a country hit hardest by COVID-19 which additionally reduced the blood donation rates. In order to address such challenges, the present study focused on the development of RBC substitutes that can be transfused to all blood types. This paper reports the development and characterization of RBC substitutes by combining the strategies of conjugated and encapsulated hemoglobin where magnetite nanoparticles would act as the carrier of hemoglobin, and liposomes would separate internal and external environments. The interactions of hemoglobin variants with bare magnetite nanoparticles were studied through molecular docking studies. Moreover, nanoparticles were synthesized, and hemoglobin was purified from blood. These components were then used to make conjugates, and it was observed that only the hemoglobin HbA1 variant was making protein corona. These conjugates were then encapsulated in liposomes to make negatively charged RBC substitutes with a size range of 1-2 μm. Results suggest that these RBC substitutes work potentially in a similar way as natural RBCs work and can be used in the time of emergency.

Intraosseous infusion of liposome-encapsulated hemoglobin (HbV) acutely prevents hemorrhagic anemia-induced lethal arrhythmias, and its efficacy persists with preventing proarrhythmic side effects in the subacute phase of severe hemodilution model.

Artificial oxygen carriers (HbV) can treat hemorrhagic shock with lethal arrhythmias (VT/VF). No reports exist on subacute HbV's effects. Acute and subacute resuscitation effects with anti-arrhythmogenesis of HbV were studied in 85% blood exchange rat model (85%-Model). Lethal 85%-Model was created by bone marrow transfusion and femoral artery bleeding in 80 SD rats in HbV-administered group (HbV-group), washed erythrocyte-administered group (wRBC-group), and 5% albumin-administered group (ALB-group). Survival rates, anti-arrhythmic efficacy by optical mapping system (OMP) with electrophysiological study (EPS) in Langendorff heart, cardiac autonomic activity by heart rate variability (HRV) and ventricular arrhythmias by 24-h electrocardiogram telemetry monitoring (24h-ECG) in awake, and left ventricular function by echocardiography (left ventricular ejection fraction [LVEF]) were measured. All rats in HbV- and wRBC-groups survived for 4 weeks, whereas no rats in ALB-group. HbV and wRBC acutely suppressed VT/VF in Langendorff heart through ameliorating action potential duration dispersion (APDd) analyzed by OMP with EPS. For subacute analysis, 50% blood exchange by 5% albumin was used (ALB-group 50). Subacute salutary effect on APDd and VT/VF inducibility was confirmed in HbV- and wRBC-groups. 24h-ECG showed that HbV and wRBC suppressed none-sustained ventricular tachycardia (NSVT) and sympathetic component of HRV (LF/HF) with preserved LVEF (HbV-group, wRBC-group vs. ALB-group 50; NSVT numbers/days, 0.5±0.3, 0.4±0.3 vs. 3.9±1.2*; LF/HF, 1.1±0.2, 0.8±0.2 vs. 3.5±1.0*; LVEF, 84±5, 83±4, vs. 77±4%*; *p<0.05). Collectively, HbV has sustained antiarrhythmic effect in subacute 85%-Model by ameliorating electrical remodeling and improving arrhythmogenic modifying factors (HRV and LVEF). These findings are useful in now continuing clinical trials of HbV.

Liposome-encapsulated hemoglobin (HbV) transfusion rescues rats undergoing progressive lethal 85% hemorrhage as a result of an anti-arrhythmogenic effect on the myocardium.

Liposome-encapsulated hemoglobin vesicles (HbV) can serve as a blood substitute with oxygen-carrying capacity comparable to that of human blood and lethal hemorrhage is associated with lethal arrhythmias. To investigate the resuscitation effect of HbV on lethal hemorrhage and anti-arrhythmogenesis, we performed optical mapping analysis (OMP) and electrophysiological study (EPS) in graded blood exchange (85% blood loss) in the rat model. We also measured cardiac autonomic activity, as assessed by heart rate variability (HRV), and changes in plasma norepinephrine and left ventricle ejection fraction (LVEF) by echocardiography. Pathological study on Connexin43 was performed. A 5% albumin (ALB group), washed rat erythrocytes (wRBC group), and HbV (HbV group) were used as a resuscitation fluid. The survival effects over 24hours were examined. All rats died in the ALB group, whereas almost all survived for 24-hours period in wRBC and HbV groups. OMP showed impaired action potential duration dispersion (APDd) in the ALB group, whereas normal APDs in HbV and wRBC groups. Lethal arrhythmias were induced by EPS in the ALB group, but not in wRBC and HbV groups. HRV indices, LVEF, Connexin43 were preserved in HbV and wRBC groups. Lethal hemorrhage causes lethal arrhythmias in the presence of impaired APDd. HbV acutely rescues lethal hemorrhage by preventing lethal arrhythmias and preserving arrhythmogenic factors.

Numerical simulations of blood cell flow in non-Newtonian fluid

We investigated how non-Newtonian viscosity behavior affects the flow characteristics of blood cells. Our findings offer insight about how shear thinning affects the dispersion of liposome-encapsulated hemoglobin and red blood cells in blood. The lattice Boltzmann method was used for fluid calculations, and the rheological properties of the non-Newtonian fluid were modeled with power-law relationships. The deformable three-dimensional red blood cell model was applied. First, we investigated the effects of shear thinning on the flow behavior of single blood cell. Simulation results indicate that shear thinning promotes the axial concentration of red blood cells. Next, varied the hematocrit to see how mutual interference between blood cells affects flow. At low hematocrit, shear thinning clearly promotes the axial concentration of red blood cells. As the hematocrit increases, in contrast, mutual interference has a greater effect, which counteracts shear thinning so the red blood cell distribution resembles the distribution within a Newtonian fluid.

Abstract 116: Acute and Chronic Anti-Arrhythmogenic Effect of Liposome-Encapsulated Hemoglobin (HbV) on the Myocardium Through Improving Myocardial Electrical Remodeling and the Arrhythmogenic Substrate in Hemorrhagic Shock-Induced Heart

Introduction: Prolonged blood pressure &lt; 40 mmHg in hemorrhagic shock (HS) causes irreversible heart dysfunction, “Shock Heart Syndrome” (SHS), which is associated with lethal arrhythmias (VT/VF). Methods: To investigate whether liposome-encapsulated human hemoglobin (HbV) is comparable to washed red blood cells (wRBCs) for improving arrhythmogenesis in SHS in either acute or chronic phase, optical mapping analysis (OMP), electrophysiological study (EPS) and pathological examinations were performed in Sprague-Dawley rat hearts, being obtained from both acute and chronic phase when each rat survived. The first, they were subjected to acute HS by withdrawing 30% of total blood volume. After HS, rats were immediately resuscitated by transfusing exactly same amount of 5% albumin (5%ALB, n=13), HbV (n=13), or wRBCs (n=13). All rats in chronic phase survived at least several weeks. After excising heart, OMP and EPS were performed in Langendorff-perfused hearts. Results: In both acute and chronic phase, OMP showed tendency for abnormal conduction and significantly impaired action potential duration dispersion (APDd) in left ventricle with 5%ALB (25±9 / 24±10 ms, Acute / Chronic phase). In contrast, myocardial conduction and APDd were substantially preserved with HbV (14±3 / 13±5 ms) and wRBCs (14±3 / 15±3 ms) as shown in Figure . Sustained VT/VF was easily provoked by burst pacing stimulus to left ventricle with 5%ALB. No VT/VF was induced with HbV and wRBCs. Pathology showed myocardial structural damage characterized by worse myocardial cell damage and Connexin43 with 5%ALB, whereas it was attenuated with HbV and wRBCs in both acute and chronic phase. Conclusions: Ventricular structural remodeling after HS causes VT/VF in the presence of APDd. Transfusion of HbV prevents acutely and chronically VT/VF, similarly to transfusion of wRBCs, by preventing chronic electrical remodeling of APDd and preserving myocardial structures in HS-induced SHS

Abstract 117: Chronic Efficacy of Liposome-Encapsulated Hemoglobin (HbV) on Rat’s Lethal 85% Hemorrhage: Intraosseus HbV Transfusion Acutely Rescues Through Anti-Arrhythmogenic Effects on Myocardium That Continues in Chronic Phase

Introduction: Liposome-encapsulated hemoglobin vesicles (HbVs) can serve as a blood substitute with human blood. Method: To investigate the resuscitation effect of HbVs on lethal hemorrhage and their efficacy for decreasing myocardial arrhythmogenesis, optical mapping analysis (OMP) and an electrophysiological study (EPS) were performed in graded 85% hemorrhage in rats in both acute and chronic phase. Six rats were resuscitated by intraosseous infusion of 5% albumin solution (ALB group), while 6 rats by washed rat erythrocytes (wRBC group) and 6 rats by HbVs (HbV group). Survival effects over 24 hours were examined in &gt; 10 rats in each group. After excising the heart, OMP and an EPS were performed. Results: All rats died in ALB group, whereas all survived subsequent 24 hours in wRBC group and HbV group. In acute phase, OMP showed impaired (prolonged) action potential duration dispersion (APDd) in left ventricle in ALB group. In contrast, myocardial APDds in left ventricle were substantially attenuated in HbV group and wRBC group. Lethal arrhythmias (ventricular tachycardia [VT] or ventricular fibrillation [VF], VT/VF) were provoked by EPS in ALB group. No VT/VF was induced in both wRBC group and HbV group. In chronic phase of two weeks after the resuscitation, OMP and an EPS were performed again in excising the heart. Anti-arrhythmic effects were confirmed by normal OMP findings (Normal APDd) and No VT/VF induced as shown in Figure. Conclusions: Lethal hemorrhage causes VT/VF in the presence of impaired APDd. Intraosseous HbVs infusion acutely rescues lethal hemorrhage through preventing lethal arrhythmias with preserved APDd. These effects were preserved in chronic phase.

Artificial oxygen carrier improves fatigue resistance in slow muscle but not in fast muscle in a rat in situ model.

The effects of liposome-encapsulated hemoglobin with high O2 affinity (h-LEH), an artificial O2 carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80Hz tetanus train, 1.5minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O2 supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate.

Abstract 193: Intraosseous Transfusion With Liposome-Encapsulated Hemoglobin (HbV) Comparably Improves Rat Survival and its Arrythmogenesis Undergoing Progressive Lethal 85% Hemorrhage With Central Venous Infusion: Possible Implication for Pre-Hospital Settings

Liposome-encapsulated hemoglobin (HbV) can serve as blood substitute in hemorrhagic shock (HS). To investigate the resuscitation effect of HbV through intraosseous transfusion (IO) on lethal hemorrhage and its efficacy on myocardial arrhythmogenesis, optical mapping analysis (OMP) and electrophysiological study (EPS) were performed in graded blood exchange up to 85% blood loss in rats. And it was compared with central venous infusion (CVI). Total 90 rats were randomly allocated into 6 subgroups as followings; through IO or CVI, gradually exchanged blood with 5% albumin (Alb-IO/CVI-groups), exchanged with washed rat erythrocyte (wRBC-IO/CVI-groups) and with HbV (HbV-IO/CVI-groups). Survival effects were examined in each six rat groups. After excising the heart, OMP and EPS were performed. All rats died in Alb-IO/CVI-groups whereas excellent and comparably survived for following &gt;48-hours in wRBC-IO/CVI-groups and HbV-IO/CVI-groups (Figure 1). OMP revealed impaired (prolonged) action potential duration (APD) dispersion in LV in Alb-IO/CVI-groups. In contrast, myocardial APD dispersions in LV were substantially attenuated in HbV-IO/CVI-groups and wRBC-IO/CVI-groups (Figure 2). Lethal arrhythmias (VT/VF) were provoked by EPS in Alb-IO/CVI-groups. No VT/VF was induced in both HbV-IO/CVI-groups and wRBC-IO/CVI-groups. Conclusions: IO vascular access could a reliable bridging method to resuscitate lethal HS by HbV. This suggests that IO with HbV resuscitation might be useful in pre-hospital settings in HS through preventing lethal arrhythmias.

Numerical simulation on effect of red blood cell motion on flow behavior of liposome-encapsulated hemoglobin

We simulated flows in microvascular vessels containing deformable red blood cells (RBCs) and liposome-encapsulated hemoglobin (LEH) particles. Blood flow was computed by the lattice Boltzmann method (LBM), and fluid–membrane interaction between the flow field and RBCs was incorporated using the immersed boundary method (IBM). The simulation results indicated that the LEH dispersion in a direction perpendicular to the main flow was largely affected by the presence of RBCs. In particular, LEH dispersion was high in the presence of rigid RBCs with low deformability. These results could provide essential information for designing LEH-mediated efficient oxygen supply to tissues.

Superior Microvascular Perfusion of Infused Liposome-Encapsulated Hemoglobin Prior to Reductions in Infarctions after Transient Focal Cerebral Ischemia

The development of cerebral infarction after transient ischemia is attributed to postischemic delayed hypoperfusion in the microvascular region. In the present study, we assessed the microvascular perfusion capacity of infused liposome-encapsulated hemoglobin (LEH) in a therapeutic approach for transient middle cerebral artery occlusion (tMCAO). Two-hour middle cerebral artery occlusion rats were immediately subjected to intra-arterial infusion of LEH (LEH group) or saline (vehicle group) or no treatment (control group), and then to recanalization. Neurological findings, infarct and edema progression, microvascular endothelial dysfunction, and inflammatory reactions were compared between the 3 groups after 24 hours of reperfusion. Microvascular perfusion in the early phase of reperfusion was evaluated by hemoglobin immunohistochemistry and transmission electron microscopy. The LEH group achieved significantly better results in all items evaluated than the other groups. Hemoglobin immunohistochemistry revealed that the number of hemoglobin-positive microvessels was significantly greater in the LEH group than in the other groups (P < .01), with microvascular perfusion being more likely in narrow microvessels (≤5 µm in diameter). An electron microscopic examination revealed that microvessels in the control group were compressed and narrowed by swollen astrocyte end-feet, whereas those in the LEH group had a less deformed appearance and contained LEH particles and erythrocytes. The results of the present study demonstrated that the infusion of LEH reduced infarctions after tMCAO with more hemoglobin-positive and less deformed microvessels at the early phase of reperfusion, suggesting that the superiority of the microvascular perfusion of LEH mediates its neuroprotective effects.

Effect of Oxygen Affinity of Liposome-Encapsulated Hemoglobin on Cerebral Ischemia and Reperfusion as Detected by Positron Emission Tomography in Nonhuman Primates.

We tested a hypothesis that liposome-encapsulated hemoglobin (LEH) with high oxygen (O2 ) affinity (h-LEH, P50 O2 = 10 mm Hg) may work better than LEH with low O2 affinity (l-LEH, P50 O2 = 40 mm Hg) in cerebral ischemia and reperfusion injury using positron emission tomography (PET) in primates undergoing middle cerebral artery (MCA) occlusion and reperfusion. Cerebral blood flow (CBF), O2 extract fraction (OEF), and cerebral metabolic rate of O2 (CMRO2 ) were successively determined by PET before ischemia, at 2 h of ischemia, immediately after reperfusion, and 3 h after reperfusion. Five minutes after MCA occlusion, 10 mL/kg of h-LEH (n = 6) was intravenously infused and compared with the results from previous data of monkeys treated with l-LEH (n = 6), empty liposome (n = 4), or saline (n = 8) as control. After the series of PET studies, the integrated area of cerebral infarction was determined histologically in 12 coronal brain slices. There was no significant difference in CBF, OEF, or CMRO2 up to 2 h of ischemia. A high CBF with a low OEF tended to be suppressed after reperfusion in LEH-treated monkeys. Three hours after reperfusion, the area of mild CMRO2 reduction (down to -30%) decreased (P < 0.05) and the area of mild CMRO2 increase (up to 30%) expanded in LEH-treated monkeys (P < 0.05) regardless of O2 affinity with no difference in the area of moderate-to-severe reduction (<-30%) or increase (<+30%) in CMRO2 compared to animals treated with empty liposome or saline. Distribution of CMRO2 reduction and histological damages showed that LEH mainly protected the cerebral cortex rather than basal ganglia where neuronal dendritic processes were severely lost. There was little difference between the animals treated with l-LEH or h-LEH both at 10 mL/kg or between treatment with empty liposome or saline. In conclusion, LEH was effective regardless of O2 affinity in preserving CMRO2 and in reducing the area of histological damage in the cerebral cortex, but not in basal ganglia, shortly after occlusion/reperfusion of MCA in monkey.

Liposome-Encapsulated Hemoglobin Accelerates Skin Wound Healing in Diabetic dB/dB Mice.

Since liposome-encapsulated hemoglobin with high O2 affinity (h-LEH, P50 O2 =10mm Hg) has been reported to accelerate skin wound healing in normal mice, it was tested in dB/dB mice with retarded wound healing, as seen in human diabetics. Two full-thickness dorsal wounds 6mm in diameter encompassed by silicone stents were created in dB/dB mice. Two days later (day 2), the animals were randomly assigned to receive intravenous h-LEH (2mL/kg, n=7) or saline (2mL/kg, n=7). The same treatment was repeated 4days after wounding (day 4), and the size of the skin lesions was analyzed by photography, surface perfusion was detected by Laser-Doppler imager, and plasma cytokines and chemokines were determined on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. The size of the ulcer compared to the skin defect or silicone stent became significantly reduced on days 4 and 7 in mice treated with h-LEH (47±8% of original size), similar to the level in wild-type mice, compared to saline-treated dB/dB mice (68±18%, P<0.01). Mice treated with h-LEH had significantly attenuated inflammatory cytokines, increased surface perfusion, and increased Ki67 expression on day 7 in accordance with the ulcer size reduction, while there was no significant difference in chemokines, histological granulation, epithelial thickness, and granulocyte infiltration detected by immunohistochemical staining in the ulcer between the treatment groups. The results suggest that h-LEH (2 mL/kg) early after wounding may accelerate skin wound healing in dB/dB mice to levels equivalent to wild-type mice probably via mechanism(s) involving reduced hypoxia, increased surface perfusion, suppressed inflammation, accelerated in situ cell proliferation and protein synthesis.

Liposome-Encapsulated Hemoglobin Improves Tumor Oxygenation as Detected by Near-Infrared Spectroscopy in Colon Carcinoma in Mice.

Liposome-encapsulated hemoglobin (LEH) with high (h-LEH, P50 O2 =10mmHg) or low O2 affinity (l-LEH, P50 O2 =40mmHg) may improve O2 delivery to sensitize tumor tissues for radiotherapy. A total of 10mL/kg of h-LEH, l-LEH, red blood cells (RBCs), or saline was infused in mice transplanted with murine colon carcinoma with near-infrared spectroscopy (NIRS) detectors set at the tumor (right leg) and intact muscle (left leg). NIRS recorded changes in the amount of oxyhemoglobin (oxyHb), deoxyhemoglobin (deoxyHb), and their sum (tHb) with the animals spontaneously breathing room air (10min), pure O2 (5min), and then back to room air. The tumor was finally excised for histological examination. In mice treated with h-LEH, tHb significantly increased compared to mice receiving other solutions. The magnitude was significantly attenuated in the tumor compared to the intact muscle under room air. Reciprocal changes in oxyHb and deoxyHb between intact muscle and tumor in response to infused solutions allowed assumption of average tissue PO2 between 30 and 40mmHg in muscle and at around 10mmHg in tumor. While O2 respiration increased oxyHb and decreased deoxyHb both in muscle and tumor, their sum or tHb consistently decreased in muscle and increased in tumor regardless of preceding infusion. Such responses were totally reversed when mice were placed under hypoxia (10% O2 ), suggesting that a lack of physiological circulatory regulation in tumor may account for heavier immunohistochemical staining for human hemoglobin in tumors of mice treated with h-LEH than with l-LEH. The results suggest that h-LEH may cause significant tumor oxygenation compared to RBC, l-LEH, or saline probably due to its nanometer size (vs. RBC) and high O2 affinity (vs. l-LEH) without increasing O2 content in the intact tissue (vs. O2 respiration) probably due to a lack of physiological circulatory regulation.

Nanovesicular liposome-encapsulated hemoglobin (LEH) prevents multi-organ injuries in a rat model of hemorrhagic shock

The goals of resuscitation in hemorrhagic shock are to correct oxygen deficit and to maintain perfusion pressure to the vital organs. We created liposome-encapsulated hemoglobin (LEH) as a nanoparticulate oxygen carrier (216±2nm) containing 7.2g/dl hemoglobin, and examined its ability to prevent the systemic manifestations of hemorrhagic shock (45% blood loss) in a rat model. We collected plasma after 6h of shock and LEH resuscitation, and determined the circulating biomarkers of systemic inflammation and functions of liver, gut, heart, and kidney. As is typical of the shock pathology, a significant increase in the plasma levels of cardiac troponin, liver function enzymes, soluble CD163 (macrophage activation), and creatinine, and the liver/gut myeloperoxidase activity was observed in the hemorrhaged rats. The plasma levels of TNF-α, IL-6, IL-1α, CINC-1, and IL-22 also increased after hemorrhagic shock. LEH administration prevented the hemorrhagic shock-induced accumulation of the markers of injury to the critical organs and pro-inflammatory cytokines. LEH also decreased the plasma levels of stress hormone corticosterone in hemorrhaged rats. Although saline also reduced the circulating corticosterone and a few other tissue injury markers, it was not as effective as LEH in restraining the plasma levels of creatinine, alanine transaminase, CD163, TNF-α, IL-6, and IL-1α. These results indicate that resuscitation with nanoparticulate LEH creates a pro-survival phenotype in hemorrhaged rats, and because of its oxygen-carrying capacity, LEH performs significantly better than saline in hemorrhagic shock.

Effect of liposome-encapsulated hemoglobin resuscitation on proteostasis in small intestinal epithelium after hemorrhagic shock.

Gut barrier dysfunction is the major trigger for multiorgan failure associated with hemorrhagic shock (HS). Although the molecular mediators responsible for this dysfunction are unclear, oxidative stress-induced disruption of proteostasis contributes to the gut pathology in HS. The objective of this study was to investigate whether resuscitation with nanoparticulate liposome-encapsulated hemoglobin (LEH) is able to restore the gut proteostatic mechanisms. Sprague-Dawley rats were recruited in four groups: control, HS, HS+LEH, and HS+saline. HS was induced by withdrawing 45% blood, and isovolemic LEH or saline was administered after 15 min of shock. The rats were euthanized at 6 h to collect plasma and ileum for measurement of the markers of oxidative stress, unfolded protein response (UPR), proteasome function, and autophagy. HS significantly increased the protein and lipid oxidation, trypsin-like proteasome activity, and plasma levels of IFNγ. These effects were prevented by LEH resuscitation. However, saline was not able to reduce protein oxidation and plasma IFNγ in hemorrhaged rats. Saline resuscitation also suppressed the markers of UPR and autophagy below the basal levels; the HS or LEH groups showed no effect on the UPR and autophagy. Histological analysis showed that LEH resuscitation significantly increased the villus height and thickness of the submucosal and muscularis layers compared with the HS and saline groups. Overall, the results showed that LEH resuscitation was effective in normalizing the indicators of proteostasis stress in ileal tissue. On the other hand, saline-resuscitated animals showed a decoupling of oxidative stress and cellular protective mechanisms.

1E12 微小血管における人工赤血球の酸素運搬過程に関する数値解析

1E12 微小血管における人工赤血球の酸素運搬過程に関する数値解析

微小血管における人工赤血球の酸素運搬過程に関する数値解析

微小血管における人工赤血球の酸素運搬過程に関する数値解析

Abstract 17692: Arrhythmogenic Mechanisms in Hemorrhagic Shock-induced Heart and Comparable Effect of Different Hemoglobin Concentration of Two Artificial Oxygen Carriers

Lethal ventricular arrhythmias (VT/VF) are serious complications after resuscitation of severe hemorrhagic shock (HS). To investigate mechanisms of arrhythmogenesis after HS and a role of two different concentration of oxygenated liposome-encapsulated human hemoglobin (LHb [Hgb=6g] or HbV [Hgb=10g]), optical mapping analysis (OMP) and electrophysiological study (EPS) were performed using rat HS model. HS was induced by withdrawing 30% blood from aorta (shock alone). Rats were resuscitated by transfusing saline, 5% albumin (5%ALB), LHb, HbV or oxygenated autologous washed red blood cells (wRBC: n=7, per group). After excising heart, OMP and EPS were performed in isolated Langendorff-perfused hearts. OMP revealed abnormal ventricular conduction delay (VCD) with impaired action potential duration dispersion (APDd) in both ventricles in shock alone, saline, and 5%ALB whereas VCD and APDd were substantially attenuated in LHb, HbV or wRBC (a). VT/VF was easily provoked by burst pacing stimulus to LV in saline and 5%ALB while few VT/VF was induced in LHb, LbV or wRBC (b). Conclusion: Ventricular electrical remodeling of abnormal VCD and APDd after HS causes VT/VF. LHb, LbV and wRBC comparably prevent VT/VF possible by attenuating ischemia-reperfusion injury in HS. Especially, even low concentration of hemoglobin in LHb is comparably effective with closely normal concentration of HbV.